Crystal of a Receptor-Ligand Complex and methods of use

ABSTRACT

The invention relates to the three-dimensional structure of a crystal of an EphB4 receptor complexed with a ligand. The three-dimensional structure of a Receptor-Ligand Complex is disclosed. The receptor-ligand crystal structure, wherein the ligand is an inhibitor molecule, is useful for providing structural information that may be integrated into drug screening and drug design processes. Thus, the invention also relates to methods for utilizing the crystal structure of the Receptor-Ligand Complex for identifying, designing, selecting, or testing inhibitors of the EphB4 receptor protein, such inhibitors being useful as therapeutics for the treatment or modulation of i) diseases; ii) disease symptoms; or iii) the effect of other physiological events mediated by the receptor.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application 60/759,167 filed Jan. 12, 2006 which is hereby incorporated by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not Applicable.

INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING

The Sequence Listing, which is a part of the present disclosure, is a written sequence listing comprising nucleotide and amino acid sequences of the present invention. The subject matter of the Sequence Listing is incorporated herein by reference in its entirety.

FIELD

The present invention relates to a three-dimensional structure of a receptor tyrosine kinase from the erythropoietin-producing hepatocellular carcinoma family of receptor tyrosine kinases (“Eph”), particularly an EphB complexed with an ephrin ligand (“Receptor-Ligand Complex”), for example an EphB4 or similar polypeptide complexed with an ephrin-B2 or analog, three-dimensional coordinates of a Receptor-Ligand Complex, models thereof, and uses of such structures and models.

INTRODUCTION

The Eph receptor tyrosine kinases and their ligands, the ephrins, regulate numerous biological processes in developing and adult tissues and have been implicated in cancer progression and in pathological forms of angiogenesis. For example, the Eph receptors and their ligands, the ephrins, play critical roles in angiogenesis during embryonic development as well as in adult tissues (Brantley-Sieders and Chen, 2004; Cheng et al., 2002; Gale and Yancopoulos, 1999; Kullander and Klein, 2002). The Eph family of receptor tyrosine kinases also regulates many other biological processes, including tissue patterning, axonal guidance, and as more recently discovered, tumorigenesis (Carmeliet and Collen, 1999; Ferrara, 1999; Pasquale, 2005; Wilkinson, 2000). Both the Eph receptor and the ephrin are membrane bound, and therefore require cell-cell contact to signal a cellular response. The interaction between Eph receptors and ephrins on adjacent cell surfaces results in multimerization and clustering of the Eph-ephrin complexes, leading to forward signaling in the Eph-expressing cell and reverse signaling in the ephrin-expressing cell. EphB4 belongs to the Eph (erythropoietin-producing hepatocellular carcinoma) family of receptor tyrosine kinases, which is divided into two subclasses, A and B, based on binding preferences and sequence conservation (Gale et al., 1996). In general, EphA receptors (EphA1-EphA10) bind to glycosyl phosphatidyl in ositol-(GPI) anchored ephrin-A ligands (ephrin-A1-ephrin-A6), while EphB receptors (EphB1-EphB6) interact with transmembrane ephrin-B ligands (ephrin-B1-ephrin-B3) (Eph Nomenclature Committee, 1997). While interactions between the Eph receptors and ephrin ligands of the same subclass are quite promiscuous, interactions between subclasses are rare. A few cross-subclass exceptions include the EphA4-ephrin-B2/B3 interactions (Takemoto et al., 2002), and the EphB2-ephrinA5 interaction, which has been characterized structurally (Himanen et al., 2004). EphB4 is unique within the Eph family in that it selectively binds ephrin-B2, while demonstrating only weak binding for both ephrin-B1 and ephrin-B3.

Eph receptors have a modular structure, consisting of an N-terminal ephrin binding domain adjacent to a cysteine-rich domain and two fibronectin type III repeats in the extracellular region. The intracellular region consists of a juxtamembrane domain, a conserved tyrosine kinase domain, a C-terminal sterile α-domain (SAM), and a PDZ binding motif. The N-terminal 180 amino acid globular domain is sufficient for high-affinity ligand binding (Himanen et al., 2001).

Several 12-amino-acid peptides that selectively bind to individual Eph receptors were recently identified by phage display (Koolpe et al., 2005; Koolpe et al., 2002; Murai et al., 2003). A number of EphB4-binding peptides could be aligned with each other and the 15 amino acid segment corresponding to the ephrin-B2 G-H loop (Koolpe et al., 2005). The TNYL EphB4-binding peptide was modified based on this alignment to include a carboxy-terminal RAW sequence. The resulting TNYL-RAW (TNYLFSPNGPIARAW; SEQ ID NO: 1) peptide is a potent antagonist of ephrin-B2 binding to EphB4, with an IC₅₀ value of ˜15 nM for the murine receptor, which is comparable to the IC₅₀ of ˜10 nM measured for ephrin-B2 (Table 1A). Interestingly, the TNYL peptide (which lacks the carboxy-terminal RAW sequence) is 10,000-fold less potent than TNYL-RAW (IC₅₀ of ˜150 μM).

Despite attempts to model the structural changes of EphB4 upon ligand binding, a detailed view of conformational arrangements of an EphB4 receptor in complex with a highly-selective ligand has remained elusive. Thus, the development of useful reagents for treatment or diagnosis of disease was hindered by lack of structural information of such a Receptor-Ligand Complex. Therefore, there is a need in the art to elucidate the three-dimensional structure and models of Receptor-Ligand Complexes, and to use such structures and models in therapeutic strategies, such as drug design.

SUMMARY

The present teachings include a method for designing a drug which interferes with an activity of an EphB4 receptor, the method comprising providing on a digital computer a three-dimensional structure of a receptor-ligand complex comprising the EphB4 receptor and at least one ligand of the EphB4 receptor, and using software comprised by the digital computer to design a chemical compound which is predicted to bind to the EphB4 receptor. The method can further comprise synthesizing the chemical compound, and evaluating the chemical compound for an ability to interfere with an activity of the EphB4 receptor.

In accordance with a further aspect, the chemical compound of the method is designed by computational interaction with reference to a three-dimensional site of the structure of the receptor-ligand complex. The three-dimensional site can include EphB4 D-E and J-K loops. The three-dimensional site can also include Leu-48, Cys-61, Leu-95, Ser-99Leu-100, Pro-101, Thr-147, Lys-149, Ala-155, and Cys-184 of human EphB4 (SEQ ID NO:26). In another aspect, the EphB4 receptor is a human EphB4 receptor.

The present teachings also include a method for determining a three-dimensional structure of a target EphB receptor-ligand complex structure comprising providing an amino acid sequence of a target EphB structure, wherein the three-dimensional structure of the target EphB structure is not known, predicting a pattern of folding of the amino acid sequence in a three-dimensional conformation using a fold recognition algorithm, and comparing the pattern of folding of the target structure amino acid sequence with the three-dimensional structure of a known EphB4 receptor-ligand complex. In certain aspects, the EphB4 receptor comprises a truncated EphB4 receptor, such as EphB4 (17-196) as set forth in SEQ ID NO: 2, and other homologs and analogs such as EphB4 (17-198) as set forth in SEQ ID NO: 3. In certain aspects, the EphB4 receptor consists essentially of an amino acid sequence as set forth in SEQ ID NO: 2 and other homologs and analogs such asEphB4 (17-198) as set forth in SEQ ID NO: 3. In certain aspects, the known receptor-ligand complex comprises a three-dimensional structure described by atomic coordinates that substantially conform to atomic coordinates set forth in Table 1. In additional aspects, the EphB4 receptor is a human EphB4 receptor.

In accordance with yet another aspect, a method is provided for generating a model of a three-dimensional structure of an EphB-ligand complex, the method comprising providing an amino acid sequence of a reference EphB4 polypeptide and an amino acid sequence of a target EphB comprised by the EphB-ligand complex, identifying structurally conserved regions shared between the reference EphB4 amino acid sequence and the target EphB amino acid sequence, and assigning atomic coordinates from the conserved regions to the target EphB-ligand complex. In certain aspects, the EphB4 polypeptide comprises a truncated EphB4 receptor, such as EphB4 (17-196) as set forth in SEQ ID NO:2, and other homologs and analogs such as EphB4 (17-198) as set forth in SEQ ID NO: 3. In certain aspects, the EphB4 polypeptide consists essentially of an amino acid sequence asset forth in SEQ ID NO: 2, and other homologs and analogs such as EphB4 (17-198) as set forth in SEQ ID NO: 3. In certain aspects, the target EphB-ligand complex comprises a three-dimensional structure described by atomic coordinates that substantially conform to atomic coordinates set forth in Table 1. In certain aspects, the reference EphB4-ligand complex comprises a three-dimensional structure described by atomic coordinates that substantially conform to atomic coordinates set forth in Table 1. In additional aspects, the EphB4 polypeptide is a human EphB4 polypeptide.

In accordance with another aspect, a method is provided for generating a model of a three-dimensional structure of an EphB receptor-ligand complex, the method comprising providing an amino acid sequence of a known EphB4 receptor in complex with at least one known ligand of the EphB4 receptor, providing an amino acid sequence of a target EphB receptor in complex with at least one target ligand of the EphB receptor, identifying structurally conserved regions shared between the known receptor-ligand complex amino acid sequence and the target receptor-ligand complex amino acid sequence, and assigning atomic coordinates of the conserved regions to the target receptor-ligand complex. In certain aspects, the EphB4 receptor comprises a truncated EphB4 receptor, such as EphB4(17-196) as set forth in SEQ ID NO: 2, and other homologs and analogs such as EphB4 (17-198) as set forth in SEQ ID NO: 3. In certain aspects, the EphB4 receptor consists essentially of an amino acid sequence as set forth in SEQ ID NO: 2 and other homologs and analogs such as EphB4 (17-198) as set forth in SEQ ID NO: 3. In certain aspects, the EphB4 receptor is a human EphB4 receptor. In additional aspects, the known receptor-ligand complex comprises a three-dimensional structure described by atomic coordinates that substantially conform to Table 1.

According to another aspect, a crystal is provided consisting essentially of an EphB4 ligand binding domain and a ligand. In certain aspects, the EphB4 ligand binding domain is a truncated EphB4 polypeptide having the sequence of SEQ ID NO: 2 and other homologs and analogs such as EphB4 (17-198) as set forth in SEQ ID NO: 3. In certain aspects, the EphB4 ligand binding domain consists essentially of EphB4 D-E and J-K loops. In certain aspects, the EphB4 ligand binding domain consists essentially of Leu-48, Cys-61, Leu-95, Ser-99 Leu-100, Pro-101, Thr-147, Lys-149, Ala-155, and Cys-184 of human EphB4(SEQ ID NO: 27). In certain aspects, the EphB4 ligand binding domain is a human EphB4 ligand binding domain. In additional aspects, the ligand is ephrin-B2. In certain aspects, the ligand comprises Phe-120, Pro-122, Leu-124, Trp-125, and Leu-127 of human ephrin-B2 (SEQ ID NO: 29). In certain aspects, the ligand comprises sequence motif NxWxL, wherein x is any amino acid. In certain aspects, the ligand is TNYL-RAW, a polypeptide having SEQ ID NO: 1. In other aspects, the ligand is a polypeptide selected from the group consisting of polypeptides having SEQ ID NO: 4 through SEQ ID NO: 26. In certain aspects, the ligand is a polypeptide having at least 50% sequence identity to a polypeptide selected from the group consisting of polypeptides having SEQ ID NO: 1 and SEQ ID NO: 4 through SEQ ID NO: 26. In certain aspects, the ligand is a polypeptide having at least 75% sequence identity to a polypeptide selected from the group consisting of polypeptides having SEQ ID NO: land SEQ ID NO: 4 through SEQ ID NO: 26. In certain other aspects, the ligand is a polypeptide having at least 90% sequence identity to a polypeptide selected from the group consisting of polypeptides having SEQ ID NO: 1 and SEQ ID NO: 4 through SEQ ID NO: 26. In additional aspects, the crystal comprises space group P41212 so as to form a unit cell of dimensions a=60.97 Å, b=60.97 Å, and c=151.7 Å.

In yet another aspect, a crystal is provided comprising a polypeptide having SEQ ID NO: 2 or 3 complexed with a ligand, wherein the crystal is sufficiently pure to determine atomic coordinates of the complex by X-ray diffraction to a resolution of about 1.65 Å. In certain aspects, the ligand comprises Phe-120, Pro-122, Leu-124, Trp-125, and Leu-127 of ephrin-B2. In certain aspects, the ligand comprises sequence motif NxWxL, wherein x is any amino acid. In certain aspects, the ligand is a polypeptide having SEQ ID NO: 1. In certain aspects, the ligand is a polypeptide selected from the group consisting of polypeptides having SEQ ID NO: 4 through SEQ ID NO: 26. In certain aspects, the ligand is a polypeptide having at least 50% sequence identity to a polypeptide selected from the group consisting of polypeptides having SEQ ID NO: 1 and SEQ ID NO: 4 through SEQ ID NO: 26. In certain aspects, the ligand is a polypeptide having at least 75% sequence identity to a polypeptide selected from the group consisting of polypeptides having SEQ ID NO: 1 and SEQ ID NO: 4 through SEQ ID NO: 26. In certain aspects, the ligand is a polypeptide having at least 90% sequence identity to a polypeptide selected from the group consisting of polypeptides having SEQ ID NO: 1 and SEQ ID NO: 4 through SEQ ID NO: 26.

In yet another aspect, a polypeptide is provided having SEQ ID NO: 2 or 3 in complex with a ligand. In certain aspects, the ligand is an ephrin. In certain aspects, the ephrin is ephrin-B2. In certain aspects, the ligand comprises Phe-120, Pro-122, Leu-124, Trp-125, and Leu-127 of ephrin-B2. In certain aspects, the ligand comprises sequence motif NxWxL, wherein x is any amino acid. In certain aspects, the ligand is a polypeptide having SEQ ID NO: 1. In certain aspects, the ligand is a polypeptide selected from the group consisting of polypeptides having SEQ ID NO: 4 through SEQ ID NO: 26. In certain aspects, the ligand is a polypeptide having at least 50% sequence identity to a polypeptide selected from the group consisting of polypeptides having SEQ ID NO: 1 and SEQ ID NO: 4 through SEQ ID NO: 26. In certain aspects, the ligand is a polypeptide having at least 75%sequence identity to a polypeptide selected from the group consisting of polypeptides having SEQ ID NO: 1 and SEQ ID NO: 4 through SEQ ID NO: 26. In certain aspects, the ligand is a polypeptide having at least 90% sequence identity to a polypeptide selected from the group consisting of polypeptides having SEQ ID NO: 1 and SEQ ID NO: 4 through SEQ ID NO: 26.

In other aspects, a therapeutic compound is provided that inhibits an activity of an EphB4 receptor, wherein the compound is selected by performing a structure based drug design using a three-dimensional structure determined for a crystal comprising an EphB4 receptor and a ligand, contacting a sample comprising the EphB4 receptor with the compound, and detecting inhibition of at least one activity of the EphB4 receptor. In certain aspects, the EphB4 is a polypeptide having SEQ ID NO: 2 or 3. In certain aspects, the EphB4 receptor is a human EphB4 receptor. In certain aspects, the ligand is a polypeptide having SEQ ID NO: 1. In certain aspects, the ligand is a polypeptide selected from the group consisting of polypeptides having SEQ ID NO: 4 through SEQ ID NO: 26. In certain aspects, the ligand is a polypeptide having at least 50% sequence identity to a polypeptide selected from the group consisting of polypeptides having SEQ ID NO: 1 and SEQ ID NO: 4 through SEQ ID NO: 26. In certain aspects, the ligand is a polypeptide having at least 75% sequence identity to a polypeptide selected from the group consisting of polypeptides having SEQ ID NO: 1 and SEQ ID NO: 4 through SEQ ID NO: 26. In certain aspects, the ligand is a polypeptide having at least 90% sequence identity to a polypeptide selected from the group consisting of polypeptides having SEQ ID NO: 1 and SEQ ID NO: 4 through SEQ ID NO: 26.

In yet another aspect, a three-dimensional computer image of the three-dimensional structure of an EphB4-ligand complex is provided wherein the structure substantially conforms to the three-dimensional coordinates listed in Table 1.

In yet another aspect, a computer-readable medium encoded with a set of three-dimensional coordinates set forth in Table 1 is provided wherein, using a graphical display software program, the three-dimensional coordinates of Table 1 create an electronic file that can be visualized on a computer capable of representing said electronic file as a three-dimensional image.

In yet another aspect, a computer-readable medium encoded with a set of three-dimensional coordinates of a three-dimensional structure which substantially conforms to the three-dimensional coordinates represented in Table 1 is provided wherein, using a graphical display software program, the set of three-dimensional coordinates create an electronic file that can be visualized on a computer capable of representing said electronic file as a three-dimensional image.

In yet another aspect, a method is provided for assaying EphB4 receptor binding to a compound, the method comprising providing an EphB4 receptor bound with a polypeptide having SEQ ID NO: 1, contacting the ligand-bound EphB4 receptor with a compound, and detecting the release of the polypeptide having SEQ ID NO: 1 from the EphB4 receptor, wherein the release of the polypeptide having SEQ ID NO: 1 is indicative of the compound binding to the EphB4 receptor. In certain aspects, the EphB4 receptor is a polypeptide having SEQ ID NO: 2 or 3. In certain aspects, the EphB4 receptor consists essentially of EphB4 D-E and J-K loops. In certain aspects, the EphB4 receptor consists essentially of Leu-48, Cys-61, Leu-95, Ser-99 Leu-100, Pro-101, Thr-147, Lys-149, Ala-155, and Cys-184 of SEQ ID NO: 27. In certain aspects, the EphB4 receptor is a human EphB4 receptor.

In another aspect, a method is provided for crystallizing an EphB4 receptor, the method comprising providing an EphB4 receptor in contact with a first polypeptide having SEQ ID NO: 1, and contacting the EphB4 receptor in contact with the first polypeptide with a second polypeptide having at least 50% sequence identity to SEQ ID NO: 1, but not identical to SEQ ID NO: 1, wherein the EphB4 receptor in contact with the first and second polypeptides forms an EphB4 receptor crystal. In certain aspects, the second polypeptide comprises at least 75% sequence identity to SEQ ID NO: 1. In certain aspects, the second polypeptide comprises at least 90% sequence identity to SEQ ID NO: 1.

In yet another aspect, a method is provided for crystallizing an EphB4 receptor, the method comprising providing an EphB4 receptor in contact with a polypeptide having SEQ ID NO: 1, and contacting the EphB4 receptor in contact with the polypeptide with a compound provided above, wherein the EphB4 receptor in contact with the polypeptide and the compound forms an EphB4 receptor crystal.

In another aspect, a composition is provided comprising EphB4 receptor, a ligand, and a compound provided above. In certain aspects, the EphB4 receptor is a polypeptide having SEQ ID NO: 2 or 3. In certain aspects, the EphB4 receptor consists essentially of EphB4 D-E and J-K loops. In certain aspects, the EphB4 receptor consists essentially of Leu-48, Cys-61, Leu-95, Ser-99 Leu-100, Pro-101, Thr-147, Lys-149, Ala-155, and Cys-184 of SEQ ID NO: 27. In certain aspects, the EphB4 receptor is a human EphB4 receptor. In certain aspects, the ligand is a polypeptide having SEQ ID NO: 1. In certain aspects, the ligand is a polypeptide selected from the group consisting of polypeptide shaving SEQ ID NO: 4 through SEQ ID NO: 26. In certain aspects, the ligand is a polypeptide having at least 50% sequence identity to a polypeptide selected from the group consisting of polypeptides having SEQ ID NO: 1 and SEQ ID NO: 4 through SEQ ID NO: 26. In certain aspects, the ligand is a polypeptide having at least 75% sequence identity to a polypeptide selected from the group consisting of polypeptides having SEQ ID NO: 1 and SEQ ID NO: 4 through SEQ ID NO: 26. In additional aspects, the ligand is a polypeptide having at least 90% sequence identity to a polypeptide selected from the group consisting of polypeptides having SEQ ID NO: 1 and SEQ ID NO: 4 through SEQ ID NO: 26.

These and other features, aspects and advantages of the present teachings will become better understood with reference to the following description, examples and appended claims.

DRAWINGS

Those of skill in the art will understand that the drawings, described below, are for illustrative purposes only. The drawings are not intended to limit the scope of the present teachings in any way.

FIG. 1. The ephrin binding domain of the EphB4 receptor in complex with an antagonistic peptide, TNYL-RAW. Upper left panel, composite image of complex; upper right panel, β-sheets and peptide; lower left panel, loops; lower right panel, α-helices. The ephrin binding domain consists of a jellyroll folding topology with 13 anti-parallel B-sheets connected by loops of varying lengths. Peptide binding orders the D-E and J-K loops, which cannot be visualized in the apo structure of the related EphB2 receptor.

FIG. 2. Superposition of the EphB4 receptor on the EphB2 receptor from the EphB2-ephrin-B2 structure (Himanen et al., 2001; Protein Data Base Accession No. 1KGY, incorporated herein by reference in its entirety). The structures are superimposed with an overall r.m.s.d. of 1.08 Å between equivalent Cα positions. The J-K loop is displaced by as much as 20 Å in EphB4 compared to EphB2.

FIG. 3. Model of the EphB4-ephrin-B2 complex. The EphB4 receptor is predicted to form interactions similar to those previously described in the EphB2-ephrin-B2 complex. Although several interactions are likely absent in the EphB4-ephrin-B2 complex compared to the EphB2-ephrin-B2 complex, the tetramer is likely to form at high EphB4 and ephrin-B2 concentrations.

FIG. 4. Stereoview of sigma-A weighted 2|F_(obs)|-|F_(calc)| electron density at 1.65 Å resolution, contoured at 1 a for the antagonistic TNYL-RAW peptide (SEQ ID NO: 1). The peptide was placed into the density after an initial round of structure refinement. The N-terminal threonine lacks clear electron density and is therefore absent from the structure.

FIG. 5. Close-up of the binding interface of a model of the EphB4-ephrinB2 (SEQ ID NO: 27, and SEQ ID NO: 29 respectively) complex. Position of the peptide is distinct from the ephrinB2 G-H loop. Both peptide and ephrin G-H loop reside within the hydrophobic binding cleft of the EphB4 receptor.

FIG. 6. EphB4-TNYL interactions. Non-covalent interactions are indicated by dashed lines.

FIG. 7. Superposition of the TNYL-RAW peptide (SEQ ID NO: 1) on the EphB4 (surface)-ephrin-B2 model. The ligand G-H loop extends into the hydrophobic binding cleft of the EphB4 receptor such that the TNYL-RAW peptide (SEQ ID NO: 1) and the ephrin-B2 G-H loop compete for the same binding site. The peptide binds distinctly within the binding cleft, inhibiting ephrin-B2 (SEQ ID NO: 29) binding at both high affinity dimerization interfaces.

DETAILED DESCRIPTION

The present invention relates to the discovery of the three-dimensional structure of a Receptor-Ligand Complex, models of such three-dimensional structures, a method of structure-based drug design using such structures, the compounds identified by such methods and the use of such compounds in therapeutic compositions. In particular, the present invention involves the crystal structure of the EphB4 receptor in complex with a highly specific antagonistic peptide at a resolution of 1.65 Å. The peptide is situated in a hydrophobic cleft of EphB4 corresponding to the cleft in EphB2 occupied by the ephrin-B2G-H loop. The crystal reveals structural features of EphB4 that, when in complex a ligand, provides a basis for antagonist design and modeling.

In particular, the structural and thermodynamic characterization of the EphB4 receptor in complex with a polypeptide having SEQ ID NO: 1 is described. The polypeptide is situated in the same hydrophobic cleft occupied by the ephrinB2 G-H loop, assuming a position distinct from this loop and preventing ligand binding interactions at two high-affinity dimerization interfaces. Although the peptide binds independently from the ephrin ligand, the interactions within the binding cleft are remarkably similar to previous complex structures, providing a stable network of interactions for binding. Further, structural analysis reveals the molecular determinants for the directed specificity of this antagonist for the EphB4 receptor, allowing the first insights into modulating pathways resulting in tumorigenesis and angiogenesis that rely on EphB4-ephrinB2 signaling.

One aspect of the present invention includes a model of a Receptor-Ligand Complex in which the model represents a three-dimensional structure of a Receptor-Ligand Complex. Another aspect of the present invention includes the three-dimensional structure of a Receptor-Ligand Complex. A three-dimensional structure of a Receptor-Ligand Complex substantially conforms with the atomic coordinates represented in Table 1. According to the present invention, the use of the term “substantially conforms” refers to at least a portion of a three-dimensional structure of a Receptor-Ligand Complex which is sufficiently spatially similar to at least a portion of a specified three-dimensional configuration of a particular set of atomic coordinates (e.g., those represented by Table 1) to allow the three-dimensional structure of a Receptor-Ligand Complex to be modeled or calculated using the particular set of atomic coordinates as a basis for determining the atomic coordinates defining the three-dimensional configuration of a Receptor-Ligand Complex.

More particularly, a structure that substantially conforms to a given set of atomic coordinates is a structure wherein at least about 50% of such structure has an average root-mean-square deviation (RMSD) of less than about 1.8 Å for the backbone atoms in secondary structure elements in each domain, and in various aspects, less than about 1.25 Å for the backbone atoms in secondary structure elements in each domain, and, in various aspects less than about 1.0 Å, in other aspects less than about 0.75 Å, less than about 0.5 Å, and, less than about 0.25 Å for the backbone atoms in secondary structure elements in each domain. In one aspect of the present invention, a structure that substantially conforms to a given set of atomic coordinates is a structure wherein at least about 75% of such structure has the recited average RMSD value, and in some aspects, at least about 90% of such structure has the recited average RMSD value, and in some aspects, about 100% of such structure has the recited average RMSD value. In particular, the above definition of “substantially conforms” can be extended to include atoms of amino acid side chains. As used herein, the phrase “common amino acid side chains” refers to amino acid side chains that are common to both the structure which substantially conforms to a given set of atomic coordinates and the structure that is actually represented by such atomic coordinates.

In another aspect of the present invention, a three-dimensional structure that substantially conforms to a given set of atomic coordinates is a structure wherein at least about 50% of the common amino acid side chains have an average RMSD of less than about 1.8 Å, and in various aspects, less than about 1.25 Å, and, in other aspects, less than about 1.0 Å, less than about 0.75 Å, less than about 0.5 Å, and less than about 0.25 Å. Inane aspect of the present invention, a structure that substantially conforms to a given set of atomic coordinates is a structure wherein at least about 75% of the common amino acid side chains have the recited average RMSD value, and in some aspects, at least about 90% of the common amino acid side chains have the recited average RMSD value, and in some aspects, about 100% of the common amino acid side chains have the recited average RMSD value.

A three-dimensional structure of a Receptor-Ligand Complex which substantially conforms to a specified set of atomic coordinates can be modeled by a suitable modeling computer program such as MODELER (A. Sali and T. L. Blundell, J. Mol. Biol., vol. 234:779-815, 1993 as implemented in the Insight II software package Insight II, available from Accelerys (San Diego, Calif.)) and those software packages listed in the Examples, using information, for example, derived from the following data: (1) the amino acid sequence of the Receptor-Ligand Complex; (2) the amino acid sequence of the related portion(s) of the protein represented by the specified set of atomic coordinates having a three-dimensional configuration; and, (3) the atomic coordinates of the specified three-dimensional configuration. A three-dimensional structure of a Receptor-Ligand Complex which substantially conforms to a specified set of atomic coordinates can also be calculated by a method such as molecular replacement, which is described in detail below.

A suitable three-dimensional structure of the Receptor-Ligand Complex for use in modeling or calculating the three-dimensional structure of another Receptor-Ligand Complex comprises the set of atomic coordinates represented in Table 1. The set of three-dimensional coordinates set forth in Table 1 is represented in standard Protein Data Bank format. The atomic coordinates have been deposited in the Protein Data Bank, having Accession No. 2BBA. According to the present invention, a Receptor-Ligand Complex has a three-dimensional structure which substantially conforms to the set of atomic coordinates represented by Table 1. As used herein, a three-dimensional structure can also be a most probable, or significant, fit with a set of atomic coordinates. According to the present invention, a most probable or significant fit refers to the fit that a particular Receptor-Ligand Complex has with a set of atomic coordinates derived from that particular Receptor-Ligand Complex. Such atomic coordinates can be derived, for example, from the crystal structure of the protein such as the coordinates determined for the Receptor-Ligand Complex structure provided herein, or from a model of the structure of the protein. For example, the three-dimensional structure of a dimeric protein, including a naturally occurring or recombinantly produced EphB4 receptor protein, substantially conforms to and is a most probable fit, or significant fit, with the atomic coordinates of Table 1. The three-dimensional crystal structure of the Receptor-Ligand Complex may comprise the atomic coordinates of Table 1. Also as an example, the three-dimensional structure of another Receptor-Ligand Complex would be understood by one of skill in the art to substantially conform to the atomic coordinates of Table 1. This definition can be applied to the other EphB4 receptor proteins in a similar manner.

For example, the structure of the EphB4 receptor establishes the general architecture of the EphB receptor family. Accordingly, in some configurations, EphB4 receptor protein sequence homology across eukaryotes can be used as a basis to predict the structure of such receptors, in particular the structure for such receptor-ligand binding sites and other conserved regions.

In various aspects of the present invention, a structure of a Receptor-Ligand Complex substantially conforms to the atomic coordinates represented in Table 1. Such values as listed in Table 1 can be interpreted by one of skill in the art. In other aspects, at three-dimensional structure of a Receptor-Ligand Complex substantially conforms to the three-dimensional coordinates represented in Table 1. In other aspects, a three-dimensional structure of a Receptor-Ligand Complex is a most probable fit with the three-dimensional coordinates represented in Table 1. Methods to determine a substantially conforming and probable fit are within the expertise of skill in the art and are described herein in the Examples section.

A Receptor-Ligand Complex that has a three-dimensional structure which substantially conforms to the atomic coordinates represented by Table 1 includes an EphB4 receptor protein having an amino acid sequence that is at least about 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence of a human EphB4 receptor protein, in particular an amino acid sequence having SEQ ID NO:27, across the full-length of the EphB4 receptor sequence. A sequence alignment program such as BLAST (available from the National Institutes of Health Internet web site http://www.ncbi.nlm.nih.gov/BLAST) may be used by one of skill in the art to compare sequences of an EphB receptor to the EphB4 receptor.

A three-dimensional structure of any Receptor-Ligand Complex can be modeled using methods generally known in the art based on information obtained from analysis of a Receptor-Ligand Complex crystal, and from other Receptor-Ligand Complex structures which are derived from a Receptor-Ligand Complex crystal. The Examples section below discloses the production of a Receptor-Ligand Complex crystal, in particular a truncated EphB4 receptor having SEQ ID NO: 2 or 3 complexed with a polypeptide having SEQ ID NO: 1, and a model of a Receptor-Ligand Complex, in particular a truncated EphB4 receptor having SEQ ID NO: 2 or 3 complexed with a polypeptide having SEQ ID NO: 1, using methods generally known in the art based on the information obtained from analysis of a Receptor-Ligand Complex crystal.

An aspect of the present invention comprises using the three-dimensional structure of a crystalline Receptor-Ligand Complex to derive the three-dimensional structure of another Receptor-Ligand Complex. Therefore, the crystalline EphB4 receptor complexed with a ligand, particularly a ligand having a sequence of SEQ ID NO: 1 or SEQ ID NOs: 4 through 26, and the three-dimensional structure of EphB4 complexed with such ligands permits one of ordinary skill in the art to now derive the three-dimensional structure, and models thereof, of any Receptor-Ligand Complex. The derivation of the structure of any Receptor-Ligand Complex can now be achieved even in the absence of having crystal I structure data for such other Receptor-Ligand Complexes, and when the crystal structure of another Receptor-Ligand Complex is available, the modeling of the three-dimensional structure of the new Receptor-Ligand Complex can be refined using the knowledge already gained from the Receptor-Ligand Complex structure.

In some configurations of the present teachings, the absence of crystal structure data for other Receptor-Ligand Complexes, the three-dimensional structures of other Receptor-Ligand Complexes can be modeled, taking into account differences in the amino acid sequence of the other Receptor-Ligand Complex. Moreover, the present invention allows for structure-based drug design of compounds which affect the activity of virtually any EphB receptor, and particularly, of EphB4.

One aspect of the present invention includes a three-dimensional structure of a Receptor-Ligand Complex, in which the atomic coordinates of the Receptor-Ligand Complex are generated by the method comprising: (a) providing an EphB receptor complexed with a ligand in crystalline form; (b) generating an electron-density map of the crystalline EphB receptor complexed with the ligand; and (c) analyzing the electron-density map to produce the atomic coordinates. For example, the structure of human EphB4 receptor in complex with a polypeptide ligand having SEQ ID NO: 1 is provided herein.

Structural Topology of the EphB4 Receptor

The crystal structure of the human EphB4 ligand binding domain (LBD) in complex with the antagonistic TNYL-RAW peptide (SEQ ID NO: 1) was refined to a 1.65 Å resolution. The structure of the EphB4 receptor is similar to the EphB2 receptor (Himanen et al., 1998), consisting of a jellyroll folding topology composed of 13 anti-parallel β-sheets(FIG. 1) arranged as a compact β-sandwich, with the concave sheet comprised of strands C, F, F′, L, H, and I, and the convex sheet comprised of strands D, E, A, M, G, K, and J (nomenclature according to Himanen et al. (Himanen et al., 1998)). Loops with a varying number of amino acids link each of these β-sheets. The corresponding loops in EphB2 have been shown to play essential roles in receptor-ligand dimerization (D-E, E-F, G-H, J-K) and tetramerization (H-I). Two conserved disulfide bridges that are strictly conserved across Eph receptor subclasses stabilize the G-H loop and the E-F/L-M loops at the top of the β-sandwich. The structure of the globular domain of EphB4 is similar to the apo, ephrin-B2-and ephrin-A5-bound EphB2 structures determined previously (FIG. 2), with root mean square deviations (RMSD) of 1.05, 1.08, and 0.94 Å over equivalent Cα positions (Himanenet al., 2004; Himanen et al., 1998; Himanen et al., 2001).

The ephrin-binding domain of human EphB4 (SEQ ID NO: 27) shares 45% sequence identity with that of human EphB2. Like the EphB2 crystals, the crystals of EphB4 in complex with the TNYL-RAW peptide (SEQ ID NO: 1) contain one molecule in the asymmetric unit. Unlike the apo EphB2 structure, however, the D-E and J-K loops are well ordered in EphB4 and form the peptide binding channel. These loops adopt novel conformations compared to the corresponding loops of the previously described EphB2-ephrin complex structures. Most notably, the J-K loop is significantly shifted in order to avoid steric interference with the peptide (FIG. 2). In fact, this loop is displaced by over 20 Å and17 Å from the furthest Cα positions in the structure of EphB2 in complex with ephrin-B2 orephrin-A5, respectively. The D-E loop is also shifted due to the presence of the antagonist peptide, deviating 2.3 Å and 3.2 Å from the structures of EphB2 in complex with ephrin-B2 and ephrin-A5, respectively. Less significant changes occur at adjacent loops involved in dimerization, due to the new position of the J-K loop, including the disulfide-stabilized G-H loop and the C-D loop, which contains a unique two amino acid insert not found in any other Eph receptors. This insert does not appear to play a role in peptide or ephrin binding and, therefore, presumably does not contribute to the ligand selectivity of EphB4.

EphB4-ephrin-B2 Interaction

Using the overall topology of the EphB4 binding cleft for comparison, the EphB4-ephrin-B2 interaction was modeled using the EphB2-ephrin-B2 structure as a starting model (FIG. 3). The ephrin-B2 G-H loop forms contacts similar to those described in the structure of the EphB2-ephrin-B2 complex. This high affinity binding interface is highly hydrophobic, and includes residues Phe-120, Pro-122, Leu-124, Trp-125, and Leu-127 of ephrin-B2. The G-H loop of ephrin-B2 is buttressed by the G-H and J-K loops of EphB4, and forms similar main chain hydrogen bonds and numerous van der Waals interactions with EphB4 as previously described in the complex with EphB2 (Himanen et al., 2004; Himanenet al., 2001). In addition, the conserved Cys-61-Cys-184 disulfide bridge of EphB4 is stabilized by Pro-122 from the conserved FSPN segment of the ephrin-B2 G-H loop. As predicted by the Pro-122 positioning in the EphB4 G-H loop, this residue assumes a position similar to that described in the complex with EphB2. The J-K loop of EphB4 shifts towards ephrin-B2 in order to maximize the binding potential between receptor and ligand, as observed in the EphB2-ephrin crystal structures. Indeed, the Eph receptor J-K loop displays remarkable flexibility and is present in a different conformation in EphB2 bound to ephrin-B2or to ephrin-A5. In addition, the J-K loop in the apo structure of EphB2 could not be visualized probably because it is disordered in the absence of a bound ligand (Himanen et al., 2004; Himanen et al., 1998; Himanen et al., 2001).

A second, lower affinity binding interface between EphB2 and ephrin-B2 has been structurally characterized (FIG. 3). This interface, which has been implicated in tetramerization, is absent in the EphB2-ephrinA5 complex, suggesting that it confers subclass binding specificity (Himanen et al., 2004; Himanen et al., 2001). The interface is framed by the H-I subclass-specificity loop. In EphB4, this loop is similar to the EphB2 H-I loop, with a maximum displacement of 2.5 Å at conserved residue Thr-39 of EphB4. Like the EphB2-ephrin-B2 low affinity interface, the EphB4-ephrin-B2 interface is dominated by hydrophobic interactions and few weak polar contacts between the receptor H-I loop and the A-A′ β strands of the ephrin. Hydrophobic interactions similar to those observed in the EphB2-ephrin-B2 complex can also be modeled between the F-G and K-L loops of EphB4and the C-D loop of ephrin-B2.

Accordingly, the present invention provides a three-dimensional structure of the EphB4 receptor protein complexed with a ligand, particularly a polypeptide having SEQ ID NO: 1, can be used to derive a model of the three-dimensional structure of another Receptor-Ligand Complex (i.e., a structure to be modeled). As used herein, a “structure” of a protein refers to the components and the manner of arrangement of the components to constitute the protein. As used herein, the term “model” refers to a representation in a tangible medium of the three-dimensional structure of a protein, polypeptide or peptide. For example, a model can be a representation of the three-dimensional structure in an electronic file, on a computer screen, on a piece of paper (i.e., on a two dimensional medium), and/or as a ball-and-stick figure. Physical three-dimensional models are tangible and include, but are not limited to, stick models and space-filling models. The phrase “imaging the model on a computer screen” refers to the ability to express (or represent) and manipulate the model on a computer screen using appropriate computer hardware and software technology known to those skilled in the art. Such technology is available from a variety of sources including, for example, Accelrys, Inc. (San Diego, Calif.). The phrase “providing a picture of the model” refers to the ability to generate a “hard copy” of the model. Hard copies include both motion and still pictures. Computer screen images and pictures of the model can be visualized in a number of formats including space-filling representations, a-carbon traces, ribbon diagrams and electron density maps.

Suitable target Receptor-Ligand Complex structures to model using a method of the present invention include any EphB receptor protein, polypeptide or peptide that is substantially structurally related to an EphB4 receptor protein complexed with a ligand. In various embodiments, a target Receptor-Ligand Complex structure that is substantially structurally related to an EphB4 receptor protein includes a target Receptor-Ligand Complex structure having an amino acid sequence that is at least about 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence of a human EphB4 receptor protein, in particular an amino acid sequence having SEQ ID NO: 27, across the full-length of the EphB4 receptor sequence when using, for example, a sequence alignment program such as BLAST (supra). In various aspects of the present invention, target Receptor-Ligand Complex structures to model include proteins comprising amino acid sequences that are at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acid sequence of a truncated EphB4 receptor, EphB4(17-196), having SEQ ID NO: 2 or EphB4 17-198, having SEQ ID NO: 3, when comparing suitable regions of the sequence, such as the amino acid sequence for an ephrin binding site of any one of the amino acid sequences, when using an alignment program such as BLAST (supra) to align the amino acid sequences.

According to the present invention, a structure can be modeled using techniques generally described by, for example, Sali, Current Opinions in Biotechnology, vol. 6, pp. 437-451, 1995, and algorithms can be implemented in program packages such as Insight II, available from Accelerys (San Diego, Calif.). Use of Insight II HOMOLOGY requires an alignment of an amino acid sequence of a known structure having a known three-dimensional structure with an amino acid sequence of a target structure to be modeled. The alignment can be a pairwise alignment or a multiple sequence alignment including other related sequences (for example, using the method generally described by Rost, Meth. Enzymol., vol. 266, pp. 525-539, 1996) to improve accuracy. Structurally conserved regions can be identified by comparing related structural features, or by examining the degree of sequence homology between the known structure and the target structure. Certain coordinates for the target structure are assigned using known structures from the known structure. Coordinates for other regions of the target structure can be generated from fragments obtained from known structures such as those found in the Protein Data Bank. Conformation of side chains of the target structure can be assigned with reference to what is sterically allowable and using a library of rotamers and their frequency of occurrence (as generally described in Ponder and Richards, J. Mol. Biol., vol. 193, pp. 775-791, 1987). The resulting model of the target structure, can be refined by molecular mechanics to ensure that the model is chemically and conformationally reasonable.

Accordingly, one embodiment of the present invention is a method to derive a model of the three-dimensional structure of a target Receptor-Ligand Complex structure the method comprising the steps of: (a) providing an amino acid sequence of a Receptor-Ligand Complex and an amino acid sequence of a target ligand-complexed EphB receptor ;(b) identifying structurally conserved regions shared between the Receptor-Ligand Complex amino acid sequence and the target ligand-complexed EphB4 receptor amino acid sequence; (c) determining atomic coordinates for the target ligand-complexed EphB4 receptor by assigning said structurally conserved regions of the target ligand-complexed EphB4 receptor to a three-dimensional structure using a three-dimensional structure of a Receptor-Ligand Complex based on atomic coordinates that substantially conform to the atomic coordinates represented in Table 1, to derive a model of the three-dimensional structure of the target ligand-complexed EphB4 receptor amino acid sequence. A model according to the present invention has been previously described herein. In one aspect, the model comprises a computer model. The method can further comprise the step of electronically simulating the structural assignments to derive a computer model of the three-dimensional structure of the target ligand-complexed EphB4 receptor amino acid sequence.

Another embodiment of the present invention is a method to derive a computer model of the three-dimensional structure of a target ligand-complexed EphB4 receptor structure for which a crystal has been produced (referred to herein as a “crystallized target structure”). A suitable method to produce such a model includes the method comprising molecular replacement. Methods of molecular replacement are generally known by those of skill in the art and are performed in a software program including, for example, XPLOR available from Accelerys (San Diego, Calif.). In various aspects, a crystallized target ligand-complexed EphB receptor structure useful in a method of molecular replacement according to the present invention has an amino acid sequence that is at least about 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of the search structure (e.g., human EphB4), when the two amino acid sequences are compared using an alignment program such as BLAST (supra). A suitable search structure of the present invention includes a Receptor-Ligand Complex having a three-dimensional structure that substantially conforms with the atomic coordinates listed in Table 1.

Another aspect of the present invention is a method to determine a three-dimensional structure of a target Receptor-Ligand Complex structure, in which the three-dimensional structure of the target Receptor-Ligand Complex structure is not known. Such a method is useful for identifying structures that are related to the three-dimensional structure of a Receptor-Ligand Complex based only on the three-dimensional structure of the target structure. For example, the present method enables identification of structures that do not have high amino acid identity with an EphB4 receptor protein but which share three-dimensional structure similarities of a ligand-complexed EphB4 receptor. In various aspects of the present invention, a method to determine a three-dimensional structure of a target Receptor-Ligand Complex structure comprises: (a) providing an amino acid sequence of a target structure, wherein the three-dimensional structure of the target structure is not known; (b) analyzing the pattern of folding of the amino acid sequence in a three-dimensional conformation by fold recognition; and (c) comparing the pattern of folding of the target structure amino acid sequence with the three-dimensional structure of a Receptor-Ligand Complex to determine the three-dimensional structure of the target structure, wherein the three-dimensional structure of the Receptor-Ligand Complex substantially conforms to the atomic coordinates represented in Table 1. For example, methods of fold recognition can include the methods generally described in Jones, Curr. Opinion Struc. Biol., vol. 7, pp. 377-387, 1997. Such folding can be analyzed based on hydrophobic and/or hydrophilic properties of a target structure.

One aspect of the present invention includes a three-dimensional computer image of the three-dimensional structure of a Receptor-Ligand Complex. In one aspect, a computer image is created to a structure which substantially conforms with the three-dimensional coordinates listed in Table 1. A computer image of the present invention can be produced using any suitable software program, including, but not limited to, Pymol available from DeLano Scientific, LLC (South San Francisco, Calif.). Suitable computer hardware useful for producing an image of the present invention is known to those of skill in the art.

Another aspect of the present invention relates to a computer-readable medium encoded with a set of three-dimensional coordinates represented in Table 1, wherein, using a graphical display software program, the three-dimensional coordinates create an electronic file that can be visualized on a computer capable of representing said electronic file as a three-dimensional image. Yet another aspect of the present invention relates to a computer-readable medium encoded with a set of three-dimensional coordinates of a three-dimensional structure which substantially conforms to the three-dimensional coordinates represented in Table 1, wherein, using a graphical display software program, the set of three-dimensional coordinates create an electronic file that can be visualized on a computer capable of representing said electronic file as a three-dimensional image. The present invention also includes a three-dimensional model of the three-dimensional structure of a target structure, such a three-dimensional model being produced by the method comprising: (a) providing an amino acid sequences of an EphB4 receptor comprised by a Receptor-Ligand Complex and an amino acid sequence of a target Receptor-Ligand Complex structure; (b) identifying structurally conserved regions shared between the EphB4 receptor amino acid sequence and the amino acid sequence comprised by the target Receptor-Ligand Complex structure; (c) determining atomic coordinates for the target Receptor-Ligand Complex by assigning the structurally conserved regions of the target Receptor-Ligand Complex to a three-dimensional structure using a three-dimensional structure of the EphB4 receptor comprised by a Receptor-Ligand Complex based on atomic coordinates that substantially conform to the atomic coordinates represented in Table 1 to derive a model of the three-dimensional structure of the target Receptor-Ligand Complex. In one aspect, the model comprises a computer model.

Any isolated EphB receptor protein can be used with the methods of the present invention. An isolated EphB receptor protein can be isolated from its natural milieu or produced using recombinant DNA technology (e.g., polymerase chain reaction (PCR) amplification, cloning) or chemical synthesis. To produce recombinant EphB receptor protein, a nucleic acid molecule encoding EphB receptor protein (e.g., SEQ ID NO: 28) can be inserted into any vector capable of delivering the nucleic acid molecule into a host cell. A nucleic acid molecule of the present invention can encode any portion of an EphB receptor protein, in various aspects a full-length EphB receptor protein, and in various aspects a soluble or truncated form of EphB4 receptor protein (i.e., a form of EphB4 receptor protein capable of being secreted by a cell that produces such protein). A suitable nucleic acid molecule to include in a recombinant vector, and particularly in a recombinant molecule, includes a nucleic acid molecule encoding a protein having the amino acid sequence represented by SEQ ID NOs: 2 or 3 and SEQ ID NO: 27.

A recombinant vector can be either RNA or DNA, either prokaryotic or eukaryotic, and typically is a virus or a plasmid. In various aspects, a nucleic acid molecule encoding an EphB4 receptor protein is inserted into a vector comprising an expression vector to form a recombinant molecule. As used herein, an expression vector is a DNA or RNA vector that is capable of transforming a host cell and of affecting expression of a specified nucleic acid molecule. Expression vectors of the present invention include any vectors that function (i.e., direct gene expression) in recombinant cells of the present invention, including in bacterial, fungal, endo parasite, insect, other animal, and plant cells.

An expression vector can be transformed into any suitable host cell to form a recombinant cell. A suitable host cell includes any cell capable of expressing a nucleic acid molecule inserted into the expression vector. For example, a prokaryotic expression vector can be transformed into a bacterial host cell. One method to isolate EphB4 receptor protein useful for producing ligand-complexed EphB4 receptor crystals includes recovery of recombinant proteins from cell cultures of recombinant cells expressing such EphB4 receptor protein.

EphB4 receptor proteins of the present invention can be purified using a variety of standard protein purification techniques, such as, but not limited to, affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, chromatofocusing and differential solubilization. In various aspects of the present invention, an EphB4 receptor protein is purified in such a manner that the protein is purified sufficiently for formation of crystals useful for obtaining information related to the three-dimensional structure of a Receptor-Ligand Complex. In some aspects, a composition of EphB4 receptor protein is about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% pure.

Another embodiment of the present invention includes a composition comprising a Receptor-Ligand Complex in a crystalline form (i.e., Receptor-Ligand Complex crystals). As used herein, the terms “crystalline Receptor-Ligand Complex” and “Receptor-Ligand Complex crystal” both refer to crystallized a Receptor-Ligand Complex and are intended to be used interchangeably. In various aspects of the present invention, a crystalline Receptor-Ligand Complex is produced using the crystal formation method described in the Examples.

In particular, the present invention includes a composition comprising EphB4 receptor complexed with a ligand in a crystalline form (i.e., ligand-complexed EphB4 crystals). As used herein, the terms “crystalline ligand-complexed EphB4” and “ligand complexed EphB4 crystal” both refer to crystallized EphB4 receptor complexed with a ligand and are intended to be used interchangeably. In various aspects of the present invention, a crystal ligand-complexed EphB4 is produced using the crystal formation method described in the Examples. In some aspects, a composition of the present invention includes ligand-complexed EphB4 molecules arranged in a crystalline manner in a space group P41212 so as to form a unit cell of dimensions a=60.97 Å, b=60.97 Å, and c=151.7 Å. A suitable crystal of the present invention provides X-ray diffraction data for determination of atomic coordinates of the ligand-complexed EphB4 to a resolution of about 1.6 Å, and in some aspects about 1.0 Å, and in other aspects at about 0.8 Å.

According to an aspect of the present invention, crystalline Receptor-Ligand Complex can be used to determine the ability of a compound of the present invention to bind to an EphB4 receptor in a manner predicted by a structure based drug design method of the present invention. In various aspects of the present invention, a Receptor-Ligand Complex crystal is soaked in a solution containing a chemical compound of the present invention. Binding of the chemical compound to the crystal is then determined by methods standard in the art.

One aspect of the present invention is a therapeutic composition. A therapeutic composition of the present invention comprises one or more therapeutic compounds. In one aspect, a therapeutic composition is provided that is capable of antagonizing the EphB4 receptor. For example, a therapeutic composition of the present invention can inhibit (i.e., prevent, block) binding of an EphB4 receptor on a cell having anEphB4 receptor (e.g., human cells) to a, e.g., ephrin-B2 or ephrin-B2 analog by interfering with the ligand binding domain of an EphB4 receptor. As used herein, the term “ligand binding domain” refers to the region of a molecule to which another molecule specifically binds.

Suitable inhibitory compounds of the present invention are compounds that interact directly with an EphB receptor protein, and in various aspects an EphB4 receptor protein or truncated EphB4 receptor protein (e.g., SEQ ID NOs: 2 or 3), thereby inhibiting the binding of an EphB4 receptor ligand, e.g., ephrin-B2, to an EphB4 receptor, by blocking the ligand binding domain of an EphB4 receptor (referred to herein as substrate analogs). An EphB4 receptor substrate analog refers to a compound that interacts with (e.g., binds to, associates with, modifies) the ligand binding domain of an EphB4 receptor. An EphB4 receptor substrate analog can, for example, comprise a chemical compound that mimics a polypeptide having SEQ ID NO: 1 or one of SEQ ID NOs: 4 through 26, or that binds specifically to the ephrin binding globular domain of an EphB4 receptor. Further examples of EphB4 receptor substrates upon which an EphB4 ligand analog can be derived are found in U.S. Patent Application No. 20040180823, incorporated herein by reference in its entirety. In various aspects, an EphB4 receptor substrate analog useful in the present invention has an amino acid sequence that is at least about 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 1 or one of SEQ ID NOs: 4 through 26.

According to the present invention, suitable therapeutic compounds of the present invention include peptides or other organic molecules, and inorganic molecules. Suitable organic molecules include small organic molecules. In various aspects, a therapeutic compound of the present invention is not harmful (e.g., toxic) to an animal when such compound is administered to an animal. Peptides refer to a class of compounds that is small in molecular weight and yields two or more amino acids upon hydrolysis. A polypeptide is comprised of two or more peptides. As used herein, a protein is comprised of one or more polypeptides. Suitable therapeutic compounds to design include peptides composed of “L” and/or “D” amino acids that are configured as normal or retro inversopeptides, peptidomimetic compounds, small organic molecules, or homo- or hetero-polymers thereof, in linear or branched configurations.

Therapeutic compounds of the present invention can be designed using structure based drug design. Structure based drug design refers to the use of computer simulation to predict a conformation of a peptide, polypeptide, protein, or conformational interaction between a peptide or polypeptide, and a therapeutic compound. In the present teachings, knowledge of the three-dimensional structure of the EphB4 ligand binding domain of an EphB4 receptor provide one of skill in the art the ability to design a therapeutic compound that binds to EphB4 receptors, is stable and results in inhibition of a biological response, such as tumorigenesis. For example, knowledge of the three-dimensional structure of the EphB4 ligand binding domain of an EphB4 receptor provides to a skilled artisan the ability to design a ligand or an analog of a ligand which can function as a substrate or ligand of an EphB4 receptor.

Suitable structures and models useful for structure-based drug design are disclosed herein. Models of target structures to use in a method of structure-based drug design include models produced by any modeling method disclosed herein, such as, for example, molecular replacement and fold recognition related methods. In some aspects of the present invention, structure based drug design can be applied to a structure of EphB4 in complex with a ligand, particularly a polypeptide having SEQ ID NO: 1, and to a model of a target EphB receptor structure.

One embodiment of the present invention is a method for designing a drug which interferes with an activity of an EphB4 receptor. In various configurations, the method comprises providing a three-dimensional structure of a Receptor-Ligand Complex comprising the EphB4 receptor and at least one ligand of the receptor; and designing a chemical compound which is predicted to bind to the EphB4 receptor. The designing can comprise using physical models, such as, for example, ball-and-stick representations of atoms and bonds, or on a digital computer equipped with molecular modeling software. In some configurations, these methods can further include synthesizing the chemical compound, and evaluating the chemical compound for ability to interfere with an activity of the EphB4 receptor.

Suitable three-dimensional structures of a Receptor-Ligand Complex and models to use with the present method are disclosed herein. According to the present invention, designing a compound can include creating a new chemical compound or searching databases of libraries of known compounds (e.g., a compound listed in a computational screening database containing three-dimensional structures of known compounds). Designing can also include simulating chemical compounds having substitute moieties at certain structural features. In some configurations, designing can include selecting a chemical compound based on a known function of the compound. In some configurations designing can comprise computational screening of one or more databases of compounds in which three-dimensional structures of the compounds are known. In these configurations, a candidate compound can be interacted virtually (e.g., docked, aligned, matched, interfaced) with the three-dimensional structure of a Receptor-Ligand Complex by computer equipped with software such as, for example, the AutoDock software package, (The Scripps Research Institute, La Jolla, Calif.) or described by Humblet and Dunbar, Animal Reports in Medicinal Chemistry, vol. 28, pp. 275-283, 1993, M Venuti, ed., Academic Press. Methods for synthesizing candidate chemical compounds are known to those of skill in the art.

Various other methods of structure-based drug design are disclosed in references such as Maulik et al., 1997, Molecular Biotechnology: Therapeutic Applications and Strategies, Wiley-Liss, Inc., which is incorporated herein by reference in its entirety. Maulik et al. disclose, for example, methods of directed design, in which the user directs the process of creating novel molecules from a fragment library of appropriately selected fragments; random design, in which the user uses a genetic or other algorithm to randomly mutate fragments and their combinations while simultaneously applying a selection criterion to evaluate the fitness of candidate ligands; and a grid-based approach in which the user calculates the interaction energy between three-dimensional structures and small fragment probes, followed by linking together of favorable probe sites.

In one aspect, a chemical compound of the present invention that binds to the ligand binding domain of a Receptor-Ligand Complex can be a chemical compound having chemical and/or stereochemical complementarity with an EphB receptor, e.g., an EphB4 receptor or ligand such as, for example, a polypeptide having SEQ ID NO: 1. in some configurations, a chemical compound that binds to the ligand binding domain an EphB4 receptor can associate with an affinity of at least about 10-6 M, at least about 10-7 M, or at least about 10-8 M.

Several sites of an EphB4 receptor can be targeted for structure based drug design. These sites include, in non-limiting example residues which contact ephrin-B2 or a polypeptide having SEQ ID NO: 1, e.g., EphB4 D-E and J-K loops; Leu-48, Cys-61, Leu-95, Ser-99 Leu-100, Pro-101, Thr-147, Lys-149, Ala-155, and Cys-184 of SEQ ID NO: 27. Conversely, the structure based drug design can be based upon the sites of the ligand which bind to the EphB receptor, e.g., Phe-120, Pro-122, Leu-124, Trp-125, and Leu-127 of ephrin-B2; and a ligand comprising a sequence motif NxWxL, wherein x is any amino acid.

TNYL-RAW Peptide Binding

The TNYL-RAW peptide (SEQ ID NO: 1) was modeled into the electron density after initial rounds of refinement using unbiased electron density from simulated annealing omit maps and |Fobs|−|Fcalc|, φcalc maps (FIG. 4). The peptide was inserted into the same cleft occupied by ephrin-B2, along the hydrophobic upper convex portion of the EphB4 receptor, which is situated on top of a β-sheet “floor” formed by β-strands D and E. In addition, loops D-E, E-F, G-H and J-K effectively buttress the peptide in the cavity, forming numerous van der Waals interactions and main chain hydrogen bonds that stabilize binding. Although the TNYL-RAW peptide (SEQ ID NO: 1) shares the FSPN sequence motif with the G-H loop of ephrin-B2 (Koolpe et al., 2005), surprisingly it assumes a distinct conformation as compared with the ephrin-B2 G-H loop bound to EphB2 (Himanen et al., 2001) (FIG. 5). The peptide has little secondary structure at the N terminus, but forms a pseudo-helix at its C-terminal end. A glycine-proline motif in the middle of the peptide induces a sharp 90° turn that angles the peptide into the upper edge of the binding cleft adjacent to the EphB4 G-H loop, where the high affinity-conferring RAW sequence binds.

The N-terminal residue of the peptide, Thr-P1, could not be modeled into the electron density map, and therefore is not depicted in the final model of the complex. The adjacent Asn-P2 is located along the plane of β-strand D of EphB4, in between the D-E and J-K loops, and forms few interactions with the receptor (FIG. 1). The position of Asn-P2 suggests that the N-terminal Thr-P1 may be disordered because it does not bind to EphB4, likely explaining its absence in the electron density map. Tyr-P3 is positioned on top of β-sheet D, forming a pseudo-sandwich between the D-E loop of the EphB4 receptor and the N-terminal end of the peptide. The hydroxyl group of the tyrosine forms a hydrogen bond with the EphB4 main chain oxygen of Ser-39 and stacking interactions with neighboring β-sheet residues (FIG. 6). Similar interactions are observed between Trp-125 of ephrin-B2 and the EphB2 D-E loop. These interactions likely play a key role in ordering this region of the bound ephrin (Himanen et al., 2004; Himanen et al., 2001).

The G-H loop of ephrin-B2 contains a conserved FSPN sequence, which plays an essential role in receptor binding and is the only sequence within the G-H loop that is also present in the TNYL-RAW peptide (SEQ ID NO: 1). Substantial hydrophobic interactions between this sequence and the G-H loop of the EphB2 receptor essentially lockephrin-B2 into the binding cleft of the receptor (Himanen et al., 2001). In the structure of the peptide in complex with EphB4, the corresponding Phe-P5 of the peptide is completely buried by the J-K loop of the receptor and by residues of the peptide, including lie-P11 and Trp-P15. This residue is situated more than 8 Å away from the equivalent phenylalanine residue in the ephrin-B2 G-H loop, and the N- to C-terminal orientation of the FSPN sequence in the ephrin and the peptide are pointed in opposite directions. Furthermore, unlike the SPN sequence of ephrin-B2 in complex with EphB2, the SPN sequence of the peptide is not buried by the hydrophobic G-H loop of EphB4, but instead is positioned along the solvent exposed surface of the receptor. The side chain of Ser-P6 forms a hydrogen bond with the main chain nitrogen of Asn-P8, which together with the intervening Pro-P7contributes to a sharp turn in the middle of the peptide. This turn positions Ile-P11 to interact with the conserved disulfide bridge in the E-F and L-M loops of EphB4 (Cys-61-Cys-184). Ile-P11 resides in the equivalent position as the conserved Pro-122 in ephrin-B2 (Pro-125 in ephrin-A5), which interacts with the corresponding disulfide bridge (Cys-60-Cys-192) in EphB2. The side chain of Ile-P11 forms a frame similar to the ephrin-B2 Pro-122 CD, CG, and CB positions, thus providing a hydrophobic backbone that stabilizes the position of the functionally important disulfide bridge in EphB4.

Alignment of a number of the EphB4-binding peptides that were identified by phage display (e.g., SEQ ID NOs: 4 through 26) revealed a conserved glycine-proline motif corresponding to a tryptophan located at the tip of the ephrin-B2 G-H loop. Although praline and tryptophan are not structurally similar, the G-P residues in the peptides were predicted to mimic the turn of the middle of the ephrin G-H loop (Koolpe et al., 2005). Surprisingly, the bend induced by the G-P motif is instead most similar to the turn present at the beginning of the ephrin-B2 G-H loop and formed by residues Phe-117, Gln-118 and Glu-119, which angle the ephrin G-H loop into the hydrophobic cleft of the Eph receptor. The G-P turn in the TNYL-RAW peptide (SEQ ID NO: 1) positions the RAW sequence into the upper edge of the EphB4 binding cleft, where Trp-P15 is effectively stabilized between the J-K and G-H loops of the receptor. Trp-P15 forms a main chain hydrogen bond with Ser-93 and hydrophobic interactions with Leu-88, Leu-93, Pro-94, Lys-142, and Phe-P5. These interactions are similar to those formed by Phe-120 in the ephrin-B2 FSPN motif. Unlike Phe-120 of ephrin-B2, however, Trp-P15 is buried within the hydrophobic binding cleft maximizing its interactions with the receptor. Arg-P13, which is also part of the peptide sequence important for high affinity binding, forms a hydrogen bond with the sidechain of Glu-43 of the receptor, and also aids in structuring the C-terminal end of the peptide by forming a side-chain to main-chain hydrogen bond with the solvent exposed Asn-P8. Together, Arg-P13 and Trp-P15 could disrupt several hydrogen bonds in the high affinity dimerization interface between EphB4 and the ephrin-B2 ligand, consistent with the antagonistic properties of the TNYLRAW peptide (SEQ ID NO: 1). Overall, the network of interactions between EphB4 and the high affinity-conferring RAW sequence is highly stable and similar to the interactions of the conserved FSPN sequence of ephrin-B2. Taken together, these data suggest that the TNYL-RAW peptide (SEQ ID NO: 1) can inhibit ephrin binding to the high affinity dimerization interface of the EphB4 ephrin-binding domain (FIG. 6).

A second region of the dimerization interface has been characterized adjacent to the high affinity dimerization interface that provides significant structural integrity for complex formation (FIG. 7), with numerous hydrogen bonds formed between receptor and ephrin from backbone-backbone, backbone-sidechain, and sidechain-sidechain contacts (Himanen et al., 2001). Several of these contacts can also be mapped onto a model of the EphB4-ephrin-B2 complex, including interactions between Asp-110 (L) and Thr-38 (R), Leu-101 (L) and Ser-47 (R) and Lys-112 (L) and Ser55 (R). The residues in this second interface of EphB4 would remain accessible to ephrin-B2 in the presence of the bound TNYL-RAW peptide (SEQ ID NO: 1). However, the protruding Arg-P13 and Asn-P8 of the peptide would sterically interfere with the positioning of β-strand G of ephrin-B2. Arg-P13 in particular extends away from the body of the peptide into the space that would be occupied by β-strand G of ephrin-B2. Therefore, the presence of the bound peptide would likely reposition the ephrin such that weak hydrogen bonds would dominate this affinity interface, making the interaction much weaker. In addition, the FSPN residues of the peptide would sterically clash with several residues at the tip of the low affinity EphB4-ephrin-B2 interface, including residues Lys-116, Phe-117, and Gln-118 of the ephrin ligand.

Thermodynamic Characterization

The molecular determinants were experimentally verified for the high affinity binding of the peptide predicted based on the crystal structure, a thermodynamic characterization of TNYL-RAW and truncated forms of this peptide using isothermal titration calorimetry (ITC). The binding of TNYL-RAW (SEQ ID NO: 1) to the human EphB4 ephrin-binding domain (amino acids 17-196; SEQ ID NO: 2) at 25° C. yields a Kd of 70 nM and a ΔHo of −14.7 kcal mol-1 (Table 1 B). As an internal control, the interaction between EphB4 (17-196) and the Eph-binding domain of human ephrin-B2 yielded a Kd of 40 nM and a βHo of +3.3 kcal mol-1. This is slightly lower than the affinity reported for the interaction between the entire mouse EphB4 extracellular domain and mouse or human ephrin-B2 (Table 1A). The existence of a third low affinity Eph-ephrin interface located outside the ephrin-binding domain provides for the difference (Smith et al., 2004).

The structural information suggests that two contact areas between EphB4 and the peptide are particularly critical for their interaction. One involves the N-terminal Tyr-P3 (TNYL) and the other the C-terminal Arg-P13 and Trp-P15 (RAW). The importance of these residues was verified by determining the Kd values for binding of peptides with N- and C-terminal truncations to human EphB4 (17-196) as measured in ITC experiments (Table 1B). Deletion of the N-terminal Thr-P1 and Asn-P2 of the peptide produced negligible changes in Kd (65-80 nM) and ΔHo. However, deletion of Tyr-P3 caused a 40-fold reduction in affinity (Kd=3.5 μM), indicating that the tyrosine is the first residue from the N-terminus of the peptide that is required for high affinity binding. The RAW sequence is predicted to play an essential role in peptide binding due to its extensive interactions with EphB4 residues in the EphB4-peptide complex structure. Truncation of this sequence indeed resulted in very weak binding (Kd>140 μM), in agreement with previous results (Koople et al., 2005), indicating that this region of the peptide provides critical binding determinants. Trp-P15 in particular is highly stabilized by both polar and hydrophobic interactions with the same region of EphB4 that is modeled to interact with the conserved FSPN sequence of ephrin-B2.

Competition studies measuring the ability of truncated forms of the TNYL-RAW peptide (SEQ ID NO: 1) to antagonize murine ephrin-B2 binding to murine EphB4 are also provided in addition to the ITC results with the human proteins. Thr-P1 and Asn-P2 do not affect the ability of TNYL-RAW to inhibit ephrin-B2 binding to EphB4 (Table 1A). In contrast, Tyr-P3 was required for efficient antagonistic properties. The IC50 for inhibition of ephrin-B2 binding to the TNYL-RAW and YL-RAW (NYLFSPNGPIARAW; SEQ IN NO: 30) peptide is approximately 40 nM and that for L-RAW (YLFSPNGPIARAW; SEQ ID NO: 31) is approximately 15 μM (Table 1A).

EphB4 is the sole member of the Eph receptor family that interacts preferentially with only one ephrin ligand, ephrin-B2, whereas it is only weakly activated by ephrin-B1 and ephrin-B3, the other two ephrins of the B subclass. EphB2, on the other hand, is activated by multiple ephrins, including one from the A subclass (Himanen et al., 2004). The overall structure of the EphB4 ephrin-binding domain is similar to that previously reported for EphB2 (Himanen et al., 2004; Himanen et al., 1998; Himanen et al., 2001). Furthermore, the overall topology of the high affinity dimerization interface is remarkably similar between the EphB2 and EphB4 structures, considering that only 42% of the residues in the EphB4 binding cleft are identical to the corresponding residues of EphB2 (Koolpe et al., 2005). However, there are important differences that could explain the higher ligands electivity of EphB4.

Several amino acid residues that make important contacts with the ephrin G-H loop in the high affinity dimerization interface of EphB2 are not conserved in EphB4. For example, Ser-194 of EphB2 is conserved in other EphB receptors but not in EphB4, where an alanine is present at the corresponding position. Therefore, EphB4 cannot form the polar interaction observed between the side chain of Ser-194 of EphB2 and the ephrin-B2 main chain oxygen of Glu-128. Furthermore, all EphB receptors have an aromatic residue at the position corresponding to Tyr-57 of EphB2. In EphB4 this position is occupied by Leu (residue 48), which cannot form a hydrogen bond with the main chain oxygen of Pro-150 of ephrin-B2 or an aromatic-aromatic interaction with Phe-113 of ephrin-B2, as observed for Tyr-57 of EphB2. Rather, Leu-48 forms only weak hydrophobic interactions with ephrin-B2. Leu-95 is present in EphB4 at the corresponding Arg-103 position of EphB2, resulting in the absence of another salt bridge that is present in the dimerization interface of EphB2 with both ephrin-B2 and ephrin-A5 (Himanen et al., 2004; Himanen et al., 2001). The presence of a leucine is unique to EphB4, because an arginine is conserved at this position in all other Eph receptors across subclasses.

Some of the differences between EphB4 and the other EphB receptors also explain the ability of the TNYL-RAW peptide (SEQ ID NO: 1) to selectively bind only to EphB4. In particular, two non-conserved amino acids of EphB4 make critical contacts with the high affinity-conferring RAW motif in the peptide. Leu-95 of EphB4 forms van der Waals interactions with both Phe-P3 and Trp-P15 of the peptide, aiding in the overall positioning of the peptide. The arginine present in the corresponding position of all other Eph receptors (Arg-103 in EphB2, see above) would result in steric clashes with both Trp-P15 and Phe-P5in the EphB4-TNYL-RAW structure. Furthermore, Thr-147 of EphB4 forms hydrophobic interactions with several residues of the peptide and aids in the overall positioning of Phe-P5from the peptide. The phenylalanine present in the corresponding position of other Eph receptors (Phe-155 in EphB2) would instead result in a steric clash with Phe-P5 of the peptide. The non-conserved Leu-48 of EphB4 also contributes to peptide binding by forming a van der Waals interaction with the tyrosine in the TNYL-RAW peptide (SEQ ID NO: 1).

Additional differences in the lower affinity tetramer interface of EphB4 and other EphB receptors may further contribute to the selectivity of EphB4 for ephrin-B2. For example, EphB4 lacks several residues involved in interactions that provide stability in the EphB2-ephrin-B2 tetrameric complex. Of particular interest is the absence of the stacking interaction between Phe-128 (EphB2) and Tyr-37 (ephrin-B2), due to the presence of an alanine (Ala-120) at the equivalent position in EphB4. An alanine at this position should result in a substantial loss of stability at the tetramer interface due not only to the absence of the stacking interaction with the ephrin aromatic residue, but also to the absence of interactions with residues Ser-139, Gly-141, and Asn-142 of ephrin-B2. Interestingly, ephrin-B1 contains a serine at the position corresponding to Tyr-37 in ephrin-B2, which is also predicted to destabilize the tetramer interface (Nikolov et al., 2005). In association with the missing aromatic in EphB4 (Phe-128) at the tetramer interface, formation of an EphB4-ephrin-B1 tetramer is highly unfavorable, providing one explanation for the weak interaction between this receptor and ligand. In addition, the presence in EphB4 of Thr-127 instead of Phe-135 of EphB2 results in the absence of the hydrophobic interaction with Glu-134 of ephrin-B2, which is not replaced by other interactions with the ephrin. Despite the weaker contacts at the tetramer interface, we have found that the EphB4 receptor can form a heterotetramer with the ephrin-B2 ligand (data not shown).

An interesting feature of the Eph receptors is the flexibility of their D-E and J-K loops, which line the high affinity ephrin binding cleft (Himanen et al., 2004; Himanen et al., 2001). These loops are disordered in the apo structure of EphB2, suggesting that a ligand is required to promote their stability. EphB2 can accommodate ephrins of both the A and B subclasses by shifting the position of the J-K loop by more than 10 Å. Furthermore in the structures of EphB2 in complex with ephrin-B2 or ephrin-A5, the J-K loop is positioned adjacent to the D-E loop, forming weak hydrophobic interactions that likely aid in the ordering of these loops. In the presence of bound TNYL-RAW peptide (SEQ ID NO: 1), the J-K loop of EphB4 is shifted by as much as 20 Å compared to the J-K loop of apo EphB2, suggesting that this region can undergo marked movements in order to accommodate a ligand. Supporting the idea that a ligand stabilizes the conformation of the Eph receptor ephrin-binding domain, EphB4 readily formed well-diffracting crystals in the presence of the TNYL-RAW peptide (SEQ ID NO: 1), whereas the apo form of the receptor did not crystallize.

The topology of the high affinity binding cleft in the complex with the TNYL-RAW peptide (SEQ ID NO: 1) can also accommodate the modeled ephrin-B2 G-H loop. Thus, despite marked differences in the primary and secondary structures of the peptide and the ephrin G-H loop, the two ligands both similarly fit in the EphB4 binding cleft. It will be interesting to model the many other EphB4-specific peptides that were identified by phage display (Koolpe et al., 2005) in order to gain information on the range of residues that can be accommodated at each position, as well as additional ligand structures that can be accommodated by the ephrin binding cleft of EphB4. Two of the peptides identified by phage display are unrelated in sequence to TNYL-RAW, but share with ephrin-B2 the sequence motif NxWxL (where x is any amino acid). Several other peptides with different sequences also appear to target the ephrin binding cleft of EphB4.

Although the precise roles of Eph receptor-ephrin bi-directional signaling in angiogenesis are incompletely understood, it is clear that the EphB4 receptor has a critical function because it is required for normal vascular development in the embryo (Gerety et al., 1999). The ability to modulate EphB4-ephrin-B2 binding will be critical to dissect the roles of these molecules in tumorigenesis and angiogenesis. Furthermore, antagonizing EphB4-ephrin-B2 binding will undoubtedly be of high therapeutic value. High affinity selective antagonists of this interaction could be used to inhibit tumor angiogenesis (Martiny-Baron et al., 2004; Noren et al., 2004) and pathological forms of angiogenesis, including inflammatory angiogenesis and the excessive retinal neovascularization that plays an important role in retinopathy of prematurity, macular degeneration, and diabetic retinopathy (Yuan et al., 2004; Zamora et al., 2005). The high resolution structure of the ephrin-binding domain of EphB4 in complex with a highly selective and potent peptide antagonist, which we report here, will allow the design of novel compounds that recapitulate the critical contacts of the peptide with EphB4 while having good pharmacokinetic properties.

Drug design strategies as specifically described above with regard to residues and regions of the ligand-complexed EphB4 receptor crystal can be similarly applied to the other EphB structures, including other EphB receptors disclosed herein. One of ordinary skill in the art, using the art recognized modeling programs and drug design methods, many of which are described herein, can modify the EphB4 design strategy according to differences in amino acid sequence. For example, this strategy can be used to design compounds which regulate a function of the EphB4 receptor in EphB receptors. In addition, one of skill in the art can use lead compound structures derived from one Eph-B receptor, such as the EphB4 receptor, and take into account differences in amino acid residues in other EphB4 receptors.

In the present method of structure-based drug design, it is not necessary to align a candidate chemical compound (i.e., a chemical compound being analyzed in, for example, a computational screening method of the present invention) to each residue in a target site. Suitable candidate chemical compounds can align to a subset of residues described for a target site. In some configurations of the present invention, a candidate chemical compound can comprise a conformation that promotes the formation of covalent or non-covalent crosslinking between the target site and the candidate chemical compound. In certain aspects, a candidate chemical compound can bind to a surface adjacent to a target site to provide an additional site of interaction in a complex. For example, when designing an antagonist (i.e., a chemical compound that inhibits the binding of a ligand to an EphB4 receptor by blocking a ligand binding domain or interface), the antagonist can be designed to bind with sufficient affinity to the binding site or to substantially prohibit a ligand from binding to a target area. It will be appreciated by one of skill in the art that it is not necessary that the complementarity between a candidate chemical compound and a target site extend overall residues specified here.

In various aspects, the design of a chemical compound possessing stereochemical complementarity can be accomplished by means of techniques that optimize, chemically or geometrically, the “fit” between a chemical compound and a target site. Such techniques are disclosed by, for example, Sheridan and Venkataraghavan, Acc. Chem Res., vol. 20, p. 322, 1987: Goodford, J. Med. Chem., vol. 27, p. 557, 1984; Beddell, Chem. Soc. Reviews, vol. 279, 1985; Hol, Angew. Chem., vol. 25, p. 767, 1986; and Verlinde and Hol, Structure, vol. 2, p. 577, 1994, each of which are incorporated by this reference herein in their entirety.

Some embodiments of the present invention for structure-based drug design comprise methods of identifying a chemical compound that complements the shape of an EphB4 receptor, particularly one that substantially conforms to the atomic coordinates of Table 1, or a structure that is related to an EphB4 receptor. Such method is referred to herein as a “geometric approach”. In a geometric approach of the present invention, the number of internal degrees of freedom (and the corresponding local minima in the molecular conformation space) can be reduced by considering only the geometric (hard-sphere) interactions of two rigid bodies, where one body (the active site) contains “pockets” or “grooves” that form binding sites for the second body (the complementing molecule, such as a ligand).

The geometric approach is described by Kuntz et al., J. Mol. Biol., vol. 161, p. 269, 1982, which is incorporated by this reference herein in its entirety. The algorithm for chemical compound design can be implemented using a software program such as AutoDock, available from The Scripps Research Institute (La Jolla, Calif.). One or more extant databases of crystallographic data (e.g., the Cambridge Structural Database System maintained by University Chemical Laboratory, Cambridge University, Lensfield Road, Cambridge CB2 IEW, U.K. or the Protein Data Bank maintained by Rutgers University) can then be searched for chemical compounds that approximate the shape thus defined. Chemical compounds identified by the geometric approach can be modified to satisfy criteria associated with chemical complementarity, such as hydrogen bonding, ionic interactions or Van der Waals interactions.

In some embodiments, a therapeutic composition of the present invention can comprise one or more therapeutic compounds. A therapeutic composition can further comprise other compounds capable of inhibiting an EphB4 receptor. A therapeutic composition of the present invention can be used to treat disease in an animal such as, for example, a human in need of treatment by administering such composition to the human. Non-limiting examples of animals to treat include mammals, reptiles and birds, companion animals, food animals, zoo animals and other economically relevant animals (e.g., racehorses and animals valued for their coats, such as minks). Additional animals to treat include dogs, cats, horses, cattle, sheep, swine, chickens, turkeys. Accordingly, in some aspects, animals to treat include humans.

A therapeutic composition of the present invention can also include an excipient, an adjuvant and/or carrier. Suitable excipients include compounds that the animal to be treated can tolerate. Examples of such excipients include water, saline, Ringer's solution, dextrose solution, Hank's solution, and other aqueous physiologically balanced salt solutions. Nonaqueous vehicles, such as fixed oils, sesame oil, ethyl oleate, or triglycerides may also be used. Other useful formulations include suspensions containing viscosity enhancing agents, such as sodium carboxymethylcellulose, sorbitol, or dextran. Excipients can also contain minor amounts of additives, such as substances that enhance isotonicity and chemical stability. Examples of buffers include phosphate buffer, bicarbonate buffer and Tris buffer, while examples of preservatives include thimerosal, o-cresol, formalin and benzyl alcohol. Standard formulations can either be liquid injectables or solids which can be taken up in a suitable liquid as a suspension or solution for injection. Thus, in a non-liquid formulation, the excipient can comprise dextrose, human serum albumin, preservatives, etc., to which sterile water or saline can be added prior to administration.

In one embodiment of the present invention, a therapeutic composition can include a carrier. Carriers include compounds that increase the half-life of a therapeutic composition in the treated animal. Suitable carriers include, but are not limited to, polymeric controlled release vehicles, biodegradable implants, liposomes, bacteria, viruses, other cells, oils, esters, and glycols.

Acceptable protocols to administer therapeutic compositions of the present invention in an effective manner include individual dose size, number of doses, frequency of dose administration, and mode of administration. Determination of such protocols can be accomplished by those skilled in the art. Modes of administration can include, but are not limited to, subcutaneous, intradermal, intravenous, intranasal, oral, transdermal, intraocular and intramuscular routes.

In yet another embodiment, a method is provided for assaying EphB4 receptor binding to a compound. The method can comprise providing an EphB4 receptor bound with a polypeptide, e.g., having SEQ ID NO: 1, followed by contacting the ligand bound EphB4 receptor with a compound. The release can be detected indicating that the compound binds to the EphB4 receptor. The EphB4 receptor can be a polypeptide having SEQ ID NO: 2 or 3. In certain embodiments, the EphB4 receptor can consist essentially of EphB4 D-E and J-K loops. The EphB4 receptor can also consist essentially of Leu-48, Cys-61, Leu-95, Ser-99 Leu-100, Pro-101, Thr-147, Lys-149, Ala-155, and Cys-184 of EphB4 (SEQ ID NO: 27). The EphB4 receptor can be a human EphB4 receptor.

In another embodiment, a method is provided for crystallizing an EphB4 receptor which includes providing an EphB4 receptor in contact with a first polypeptide having SEQ ID NO: 1, followed by contacting the EphB4 receptor in contact with the first polypeptide with a second polypeptide having at least 50% sequence identity to SEQ ID NO:1, but not identical to SEQ ID NO: 1, wherein the EphB4 receptor in contact with the first and second polypeptides forms an EphB4 receptor crystal. The second polypeptide can comprise at least 75% sequence identity to SEQ ID NO: 1, and in certain embodiments, at least 90% sequence identity to SEQ ID NO: 1.

In yet another embodiment, a method is provided for crystallizing an EphB4 receptor which includes providing an EphB4 receptor in contact with a polypeptide having SEQ ID NO: 1, followed by contacting the EphB4 receptor in contact with the polypeptide with a therapeutic compound as provided above, wherein the EphB4 receptor in contact with the polypeptide and the compound forms an EphB4 receptor crystal.

In another embodiment, a composition is provided comprising EphB4 receptor, a ligand, and a therapeutic compound as provided above. The EphB4 receptor can be a polypeptide having SEQ ID NO: 2 or 3. The EphB4 receptor can also consist essentially of EphB4 D-E and J-K loops or Leu-48, Cys-61, Leu-95, Ser-99 Leu-100, Pro-101, Thr-147, Lys-149, Ala-155, and Cys-184 of SEQ ID NO: 27. In certain embodiments, the EphB4 receptor can be a human EphB4 receptor.

In certain embodiments, the ligand can be a polypeptide having SEQ ID NO: 1 or polypeptides having SEQ ID NO: 4 through SEQ ID NO: 26. In other embodiments, the ligand can be a polypeptide having at least 50%, 75% or 90% sequence identity to a polypeptide selected from the group consisting of polypeptides having SEQ ID NO: 1 and SEQ ID NO: 4 through SEQ ID NO: 26. TABLE 1 Protein Databank Coordinates of Eph4 Receptor-Complexed TNYL-RAW HEADER ----- XX-XXX-XX xxxx COMPND --- REMARK 3 REMARK 3 REFINEMENT. REMARK 3 PROGRAM: REFMAC 5.2.0005 REMARK 3 AUTHORS: MURSHUDOV, VAGIN, DODSON REMARK 3 REMARK 3 REFINEMENT TARGET: MAXIMUM LIKELIHOOD REMARK 3 REMARK 3 DATA USED IN REFINEMENT. REMARK 3 RESOLUTION RANGE HIGH (ANGSTROMS): 1.65 REMARK 3 RESOLUTION RANGE LOW (ANGSTROMS): 56.61 REMARK 3 DATA CUTOFF (SIGMA(F)): NONE REMARK 3 COMPLETENESS FOR RANGE (%): 99.92 REMARK 3 NUMBER OF REFLECTIONS: 31786 REMARK 3 REMARK 3 FIT TO DATA USED IN REFINEMENT. REMARK 3 CROSS-VALIDATION METHOD: THROUGHOUT REMARK 3 FREE R VALUE TEST SET SELECTION: RANDOM REMARK 3 R VALUE (WORKING + TEST SET): 0.16824 REMARK 3 R VALUE (WORKING SET): 0.16622 REMARK 3 FREE R VALUE: 0.18595 REMARK 3 FREE R VALUE TEST SET SIZE (%): 10.0 REMARK 3 FREE R VALUE TEST SET COUNT: 3533 REMARK 3 REMARK 3 FIT IN THE HIGHEST RESOLUTION BIN. REMARK 3 TOTAL NUMBER OF BINS USED: 20 REMARK 3 BIN RESOLUTION RANGE HIGH: 1.650 REMARK 3 BIN RESOLUTION RANGE LOW: 1.693 REMARK 3 REFLECTION IN BIN (WORKING SET): 2286 REMARK 3 BIN COMPLETENESS (WORKING + TEST) (%): 98.94 REMARK 3 BIN R VALUE (WORKING SET): 0.156 REMARK 3 BIN FREE R VALUE SET COUNT: 243 REMARK 3 BIN FREE R VALUE: 0.195 REMARK 3 REMARK 3 NUMBER OF NON-HYDROGEN ATOMS USED IN REFINEMENT. REMARK 3 ALL ATOMS: 1843 REMARK 3 REMARK 3 B VALUES. REMARK 3 FROM WILSON PLOT (A**2): NULL REMARK 3 MEAN B VALUE (OVERALL, A**2): 11.757 REMARK 3 OVERALL ANISOTROPIC B VALUE. REMARK 3 B11 (A**2): 0.21 REMARK 3 B22 (A**2): 0.21 REMARK 3 B33 (A**2): −0.42 REMARK 3 B12 (A**2): 0.00 REMARK 3 B13 (A**2): 0.00 REMARK 3 B23 (A**2): 0.00 REMARK 3 REMARK 3 ESTIMATED OVERALL COORDINATE ERROR. REMARK 3 ESU BASED ON R VALUE (A): 0.078 REMARK 3 ESU BASED ON FREE R VALUE (A): 0.077 REMARK 3 ESU BASED ON MAXIMUM LIKELIHOOD (A): 0.041 REMARK 3 ESU FOR B VALUES BASED ON MAXIMUM LIKELIHOOD (A**2): 2.276 REMARK 3 REMARK 3 CORRELATION COEFFICIENTS. REMARK 3 CORRELATION COEFFICIENTS FO-FC: 0.958 REMARK 3 CORRELATION COEFFICIENTS FO-FC FREE: 0.952 REMARK 3 REMARK 3 RMS DEVIATIONS FROM IDEAL VALUES COUNT RMS WEIGHT REMARK 3 BOND LENGTHS REFINED ATOMS (A): 1667; 0.017; 0.021 REMARK 3 BOND LENGTHS OTHERS (A): 1438; 0.002; 0.020 REMARK 3 BOND ANGLES REFINED ATOMS (DEGREES): 2282; 1.801; 1.959 REMARK 3 BOND ANGLES OTHERS (DEGREES): 3327; 0.894; 3.000 REMARK 3 TORSION ANGLES, PERIOD 1 (DEGREES): 201; 7.448; 5.000 REMARK 3 TORSION ANGLES, PERIOD 2 (DEGREES): 72; 31.759; 23.056 REMARK 3 TORSION ANGLES, PERIOD 3 (DEGREES): 242; 10.377; 15.000 REMARK 3 TORSION ANGLES, PERIOD 4 (DEGREES): 9; 16.603; 15.000 REMARK 3 CHIRAL-CENTER RESTRAINTS (A**3): 246; 0.117; 0.200 REMARK 3 GENERAL PLANES REFINED ATOMS (A): 1823; 0.010; 0.020 REMARK 3 GENERAL PLANES OTHERS (A): 348; 0.001; 0.020 REMARK 3 NON-BONDED CONTACTS REFINED ATOMS (A): 244; 0.277; 0.200 REMARK 3 NON-BONDED CONTACTS OTHERS (A): 1343; 0.212; 0.200 REMARK 3 NON-BONDED TORSION REFINED ATOMS (A): 744; 0.182; 0.200 REMARK 3 NON-BONDED TORSION OTHERS (A): 881; 0.88; 0.200 REMARK 3 H-BOND (X . . . Y) REFINED ATOMS (A): 144; 0.156; 0.200 REMARK 3 H-BOND (X . . . Y) OTHERS (A): 1; 0.026; 0.200 REMARK 3 SYMMETRY VDW REFINED ATOMS (A): 23; 0.705; 0.200 REMARK 3 SYMMETRY VDW OTHERS (A): 86; 0.356; 0.200 REMARK 3 SYMMETRY H-BOND REFINED ATOMS (A): 16; 0.630; 0.200 REMARK 3 REMARK 3 ISOTROPIC THERMAL FACTOR RESTRAINTS. COUNT RMS WEIGHT REMARK 3 MAIN-CHAIN BOND REFINED ATOMS (A**2): 1080; 1.274; 1.500 REMARK 3 MAIN-CHAIN BOND OTHER ATOMS (A**2): 407; 0.336; 1.500 REMARK 3 MAIN-CHAIN ANGLE REFINED ATOMS (A**2): 1609; 1.704; 2.000 REMARK 3 SIDE-CHAIN BOND REFINED ATOMS (A**2): 750; 2.842; 3.000 REMARK 3 SIDE-CHAIN ANGLE REFINED ATOMS (A**2): 672; 4.040; 4.500; REMARK 3 REMARK 3 NCS RESTRAINTS STATISTICS REMARK 3 NUMBER OF NCS GROUPS: NULL REMARK 3 REMARK 3 REMARK 3 TLS DETAILS REMARK 3 NUMBER OF TLS GROUPS: 2 REMARK 3 ATOM RECORD CONTAINS RESIDUAL B FACTORS ONLY REMARK 3 REMARK 3 TLS GROUP: 1 REMARK 3 NUMBER OF COMPONENTS GROUP: 1 REMARK 3 COMPONENTS C SSSEQI TO C SSSEQI REMARK 3 RESIDUE RANGE: A 12 A 196 REMARK 3 ORIGIN FOR THE GROUP (A): 24.6062 11.8279 51.1982 REMARK 3 T TENSOR REMARK 3 T11: −0.0492 T22: −0.0141 REMARK 3 T33: −0.0334 T12: −0.0003 REMARK 3 T13: 0.0030 T23: −0.0021 REMARK 3 L TENSOR REMARK 3 L11: 1.2428 L22: 0.2699 REMARK 3 L33: 0.4593 L12: −0.0754 REMARK 3 L13: −0.3122 L23: 0.0783 REMARK 3 S TENSOR REMARK 3 S11: 0.0019 S12: 0.0337 S13: −0.0331 REMARK 3 S21: −0.0179 S22: −0.0002 S23: −0.0058 REMARK 3 S31: −0.0101 S32: −0.325 S33: −0.0018 REMARK 3 REMARK 3 TLS GROUP: 2 REMARK 3 NUMBER OF COMPONENTS GROUP: 1 REMARK 3 COMPONENTS C SSSEQI TO C SSSEQI REMARK 3 RESIDUE RANGE: P 251 P 264 REMARK 3 ORIGIN FOR THE GROUP (A): 31.4082 0.9166 39.8199 REMARK 3 T TENSOR REMARK 3 T11: −0.439 T22: −0.0267 REMARK 3 T33: −0.0328 T12: 0.0096 REMARK 3 T13: 0.0511 T23: −0.0513 REMARK 3 L TENSOR REMARK 3 L11: 5.1441 L22: 2.1674 REMARK 3 L33: 7.0541 L12: 1.1502 REMARK 3 L13: 3.4336 L23: −0.5897 REMARK 3 S TENSOR REMARK 3 S11: −0.1929 S12: 0.1435 S13: −0.2481 REMARK 3 S21: −0.1624 S22: 0.0877 S23: −0.1382 REMARK 3 S31: 0.2811 S32: 0.1920 S33: 0.1053 REMARK 3 REMARK 3 REMARK 3 BULK SOLVENT MODELLING. REMARK 3 METHOD USED: MASK REMARK 3 PARAMETERS FOR MASK CALCUALTION REMARK 3 VDW PROBE RADIUS: 1.20 REMARK 3 ION PROBE RADIUS: 0.80 REMARK 3 SHRINKAGE RADIUS: 0.80 REMARK 3 REMARK 3 OTHER REFINEMENT REMARKS: REMARK 3 HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS REMARK 3 SSBOND 1 CYS A 61 CYS A 184 SSBOND 2 CYS A 97 CYS A 107 CISPEP 1 PHE A 35 PRO A 36 0.00 CISPEP 2 THR A 127 PRO A 128 0.00 CISPEP 3 ASN A 133 PRO A 134 0.00 CISPEP 4 GLY A 167 PRO A 168 0.00 CRYST1 60.972 60.972 151.681 90.00 90.00 90.00 P 41 21 2 SCALE1 0.016401 0.000000 0.000000 0.00000 SCALE2 0.000000 0.016401 0.000000 0.00000 SCALE3 0.000000 0.000000 0.006593 0.00000 ATOM 1 N HIS A 12 33.704 21.776 75.467 1.00 23.03 N ATOM 2 CA HIS A 12 34.487 22.989 75.312 1.00 20.91 C ATOM 4 CB HIS A 12 33.719 24.090 74.745 1.00 20.12 C ATOM 7 CG HIS A 12 34.117 24.583 73.396 1.00 2.00 C ATOM 8 ND1 HIS A 12 33.142 24.401 72.432 1.00 18.31 N ATOM 10 CE1 HIS A 12 33.487 25.097 71.361 1.00 22.05 C ATOM 12 NE2 HIS A 12 34.473 25.876 71.733 1.00 17.59 N ATOM 14 CD2 HIS A 12 34.710 25.639 73.059 1.00 2.00 C ATOM 16 C HIS A 12 34.821 23.565 76.638 1.00 23.03 C ATOM 17 O HIS A 12 35.059 24.763 76.648 1.00 26.11 O ATOM 21 N HIS A 13 34.796 22.824 77.760 1.00 24.22 N ATOM 22 CA HIS A 13 35.792 23.137 78.752 1.00 21.79 C ATOM 24 CB HIS A 13 35.635 22.485 80.143 1.00 24.26 C ATOM 27 CG HIS A 13 36.377 21.203 80.304 1.00 27.78 C ATOM 28 ND1 HIS A 13 35.792 19.975 80.068 1.00 33.44 N ATOM 30 CE1 HIS A 13 36.686 19.023 80.270 1.00 32.20 C ATOM 32 NE2 HIS A 13 37.833 19.593 80.595 1.00 31.56 N ATOM 34 CD2 HIS A 13 37.666 20.956 80.618 1.00 28.51 C ATOM 36 C HIS A 13 37.116 22.806 78.029 1.00 19.23 C ATOM 37 O HIS A 13 38.060 23.526 78.235 1.00 18.18 O ATOM 39 N HIS A 14 37.153 21.811 77.114 1.00 16.79 N ATOM 40 CA HIS A 14 38.201 21.799 76.068 1.00 16.44 C ATOM 42 CB HIS A 14 39.054 20.546 76.115 1.00 16.82 C ATOM 45 CG HIS A 14 39.769 20.380 77.409 1.00 18.04 C ATOM 46 ND1 HIS A 14 39.709 19.220 78.150 1.00 22.52 N ATOM 48 CE1 HIS A 14 40.403 19.384 79.261 1.00 20.48 C ATOM 50 NE2 HIS A 14 40.900 20.609 79.272 1.00 22.11 N ATOM 52 CD2 HIS A 14 40.530 21.245 78.113 1.00 19.51 C ATOM 54 C HIS A 14 37.569 21.900 74.708 1.00 13.95 C ATOM 55 O HIS A 14 36.698 21.131 74.396 1.00 13.43 O ATOM 57 N HIS A 15 38.008 22.870 73.924 1.00 11.83 N ATOM 58 CA HIS A 15 37.403 23.182 72.623 1.00 11.43 C ATOM 60 CB HIS A 15 37.811 24.582 72.208 1.00 12.51 C ATOM 63 CG HIS A 15 37.613 25.585 73.298 1.00 15.38 C ATOM 64 ND1 HIS A 15 36.373 26.065 73.622 1.00 18.20 N ATOM 66 CE1 HIS A 15 36.475 26.923 74.627 1.00 15.56 C ATOM 68 NE2 HIS A 15 37.744 26.993 74.977 1.00 16.84 N ATOM 70 CD2 HIS A 15 38.471 26.148 74.173 1.00 14.49 C ATOM 72 C HIS A 15 37.823 22.178 71.572 1.00 10.00 C ATOM 73 O HIS A 15 39.029 21.913 71.367 1.00 9.89 O ATOM 75 N HIS A 16 36.829 21.605 70.889 1.00 9.16 N ATOM 76 CA HIS A 16 37.126 20.594 69.895 1.00 8.66 C ATOM 78 CB HIS A 16 37.485 19.278 70.572 1.00 9.36 C ATOM 81 CG HIS A 16 36.378 18.666 71.384 1.00 12.06 C ATOM 82 ND1 HIS A 16 36.052 19.115 72.645 1.00 13.77 N ATOM 84 CE1 HIS A 16 35.077 18.366 73.134 1.00 16.21 C ATOM 86 NE2 HIS A 16 34.785 17.427 72.245 1.00 14.78 N ATOM 88 CD2 HIS A 16 35.606 17.579 71.155 1.00 13.75 C ATOM 90 C HIS A 16 35.957 20.379 68.944 1.00 8.83 C ATOM 91 O HIS A 16 34.834 20.813 69.251 1.00 9.76 O ATOM 93 N GLU A 17 36.189 19.627 67.866 1.00 7.24 N ATOM 94 CA GLU A 17 35.066 19.171 67.022 1.00 7.03 C ATOM 96 CB GLU A 17 35.446 19.014 65.549 1.00 7.82 C ATOM 99 CG GLU A 17 35.649 20.369 64.832 1.00 6.61 C ATOM 102 CD GLU A 17 36.201 20.232 63.446 1.00 8.29 C ATOM 103 OE1 GLU A 17 36.658 19.131 63.080 1.00 8.96 O ATOM 104 OE2 GLU A 17 36.147 21.259 62.691 1.00 8.78 O ATOM 105 C GLU A 17 34.638 17.804 67.554 1.00 7.32 C ATOM 106 O GLU A 17 35.474 16.991 67.936 1.00 8.43 O ATOM 108 N GLU A 18 33.330 17.584 67.549 1.00 7.30 N ATOM 109 CA GLU A 18 32.706 16.372 68.110 1.00 9.43 C ATOM 111 CB GLU A 18 31.807 16.800 69.239 1.00 11.29 C ATOM 114 CG GLU A 18 31.129 15.663 69.960 1.00 13.90 C ATOM 117 CD GLU A 18 32.079 14.901 70.885 1.00 22.21 C ATOM 118 OE1 GLU A 18 33.301 15.167 70.928 1.00 22.48 O ATOM 119 OE2 GLU A 18 31.574 13.976 71.533 1.00 24.63 O ATOM 120 C GLU A 18 31.873 15.714 66.996 1.00 8.52 C ATOM 121 O GLU A 18 31.031 16.361 66.335 1.00 7.95 O ATOM 123 N THR A 19 32.122 14.426 66.790 1.00 7.26 N ATOM 124 CA THR A 19 31.423 13.651 65.774 1.00 8.12 C ATOM 126 CB THR A 19 32.216 12.399 65.428 1.00 7.71 C ATOM 128 OG1 THR A 19 33.491 12.739 64.803 1.00 8.16 O ATOM 130 CG2 THR A 19 31.450 11.491 64.534 1.00 7.98 C ATOM 134 C THR A 19 29.990 13.249 66.195 1.00 6.76 C ATOM 135 O THR A 19 29.800 12.787 67.352 1.00 8.39 O ATOM 137 N LEU A 20 29.037 13.430 65.273 1.00 7.50 N ATOM 138 CA LEU A 20 27.680 12.910 65.412 1.00 8.69 C ATOM 140 CB LEU A 20 26.663 13.989 65.076 1.00 7.93 C ATOM 143 CG LEU A 20 26.630 15.185 66.010 1.00 8.84 C ATOM 145 CD1 LEU A 20 25.765 16.318 65.438 1.00 11.10 C ATOM 149 CD2 LEU A 20 26.188 14.811 67.481 1.00 9.30 C ATOM 153 C LEU A 20 27.444 11.629 64.566 1.00 9.13 C ATOM 154 O LEU A 20 26.607 10.792 64.953 1.00 10.80 O ATOM 156 N LEU A 21 28.130 11.502 63.445 1.00 8.78 N ATOM 157 CA LEU A 21 28.000 10.372 62.568 1.00 8.95 C ATOM 159 CB LEU A 21 26.719 10.483 61.749 1.00 8.65 C ATOM 162 CG LEU A 21 26.479 9.488 60.622 1.00 11.72 C ATOM 164 CD1 LEU A 21 26.560 8.089 61.045 1.00 18.08 C ATOM 168 CD2 LEU A 21 25.080 9.799 60.008 1.00 14.08 C ATOM 172 C LEU A 21 29.220 10.319 61.680 1.00 8.86 C ATOM 173 O LEU A 21 29.599 11.317 61.146 1.00 9.99 O ATOM 175 N ASN A 22 29.816 9.139 61.512 1.00 8.68 N ATOM 176 CA ASN A 22 30.886 8.943 60.530 1.00 7.18 C ATOM 178 CB ASN A 22 32.262 9.048 61.208 1.00 7.76 C ATOM 181 CG ASN A 22 33.411 8.909 60.207 1.00 7.95 C ATOM 182 OD1 ASN A 22 33.209 8.470 59.053 1.00 9.79 O ATOM 183 ND2 ASN A 22 34.620 9.287 60.638 1.00 11.49 N ATOM 186 C ASN A 22 30.694 7.591 59.869 1.00 8.44 C ATOM 187 O ASN A 22 30.911 6.555 60.494 1.00 8.66 O ATOM 189 N THR A 23 30.196 7.619 58.647 1.00 8.29 N ATOM 190 CA THR A 23 29.883 6.375 57.972 1.00 8.29 C ATOM 192 CB THR A 23 29.277 6.589 56.556 1.00 9.39 C ATOM 194 OG1 THR A 23 30.222 7.277 55.734 1.00 9.28 O ATOM 196 CG2 THR A 23 27.976 7.304 56.639 1.00 9.26 C ATOM 200 C THR A 23 31.078 5.392 57.853 1.00 8.69 C ATOM 201 O THR A 23 30.879 4.188 57.814 1.00 9.41 O ATOM 203 N LYS A 24 32.300 5.912 57.767 1.00 8.61 N ATOM 204 CA LYS A 24 33.504 5.081 57.613 1.00 9.10 C ATOM 206 CB LYS A 24 34.728 5.936 57.267 1.00 8.84 C ATOM 209 CG LYS A 24 34.635 6.631 55.911 1.00 10.31 C ATOM 212 CD LYS A 24 35.793 7.539 55.642 1.00 12.72 C ATOM 215 CE LYS A 24 35.586 8.923 56.230 1.00 19.96 C ATOM 218 NZ LYS A 24 36.870 9.639 56.390 1.00 21.45 N ATOM 222 C LYS A 24 33.767 4.202 58.849 1.00 9.30 C ATOM 223 O LYS A 24 34.583 3.279 58.791 1.00 10.78 O ATOM 225 N LEU A 25 33.118 4.548 59.973 1.00 7.98 N ATOM 226 CA LEU A 25 33.261 3.773 61.196 1.00 9.56 C ATOM 228 CB LEU A 25 33.431 4.698 62.394 1.00 8.59 C ATOM 231 CG LEU A 25 34.666 5.591 62.340 1.00 11.50 C ATOM 233 CD1 LEU A 25 34.608 6.552 63.511 1.00 11.62 C ATOM 237 CD2 LEU A 25 35.915 4.801 62.362 1.00 14.34 C ATOM 241 C LEU A 25 32.158 2.717 61.406 1.00 9.15 C ATOM 242 O LEU A 25 32.218 1.955 62.379 1.00 12.15 O ATOM 244 N GLU A 26 31.167 2.664 60.531 1.00 8.87 N ATOM 245 CA GLU A 26 30.142 1.641 60.585 1.00 10.20 C ATOM 247 CB GLU A 26 29.022 1.940 59.577 1.00 10.65 C ATOM 250 CG GLU A 26 27.963 0.852 59.519 1.00 11.96 C ATOM 253 CD GLU A 26 27.288 0.604 60.839 1.00 17.08 C ATOM 254 OE1 GLU A 26 26.798 1.600 61.451 1.00 20.71 O ATOM 255 OE2 GLU A 26 27.230 −0.595 61.279 1.00 16.36 O ATOM 256 C GLU A 26 30.749 0.284 60.259 1.00 11.21 C ATOM 257 O GLU A 26 31.342 0.123 59.217 1.00 13.21 O ATOM 259 N THR A 27 30.579 −0.679 61.156 1.00 14.03 N ATOM 260 CA THR A 27 31.199 −1.991 60.979 1.00 16.39 C ATOM 262 CB THR A 27 31.779 −2.520 62.316 1.00 17.17 C ATOM 264 OG1 THR A 27 30.725 −2.729 63.251 1.00 18.71 O ATOM 266 CG2 THR A 27 32.793 −1.537 62.898 1.00 18.99 C ATOM 270 C THR A 27 30.254 −3.045 60.378 1.00 17.58 C ATOM 271 O THR A 27 30.728 −4.107 59.928 1.00 21.24 O ATOM 273 N ALA A 28 28.960 −2.766 60.383 1.00 15.57 N ATOM 274 CA ALA A 28 27.977 −3.671 59.830 1.00 16.89 C ATOM 276 CB ALA A 28 26.981 −4.129 60.889 1.00 16.73 C ATOM 280 C ALA A 28 27.369 −2.859 58.721 1.00 17.49 C ATOM 281 O ALA A 28 28.145 −2.160 58.022 1.00 20.58 O ATOM 283 N ASP A 29 26.063 −2.917 58.523 1.00 15.97 N ATOM 284 CA ASP A 29 25.446 −2.197 57.420 1.00 17.34 C ATOM 286 CB ASP A 29 24.522 −3.136 56.640 1.00 19.00 C ATOM 289 CG ASP A 29 25.301 −4.257 55.860 1.00 22.70 C ATOM 290 OD1 ASP A 29 26.556 −4.303 55.845 1.00 25.94 O ATOM 291 OD2 ASP A 29 24.626 −5.102 55.234 1.00 33.57 O ATOM 292 C ASP A 29 24.739 −0.927 57.917 1.00 15.71 C ATOM 293 O ASP A 29 24.199 −0.841 59.071 1.00 16.45 O ATOM 295 N LEU A 30 24.729 0.099 57.067 1.00 13.49 N ATOM 296 CA LEU A 30 24.132 1.369 57.449 1.00 13.28 C ATOM 298 CB LEU A 30 24.507 2.446 56.436 1.00 12.20 C ATOM 301 CG LEU A 30 25.968 2.938 56.482 1.00 11.60 C ATOM 303 CD1 LEU A 30 26.311 3.826 55.253 1.00 10.66 C ATOM 307 CD2 LEU A 30 26.230 3.709 57.775 1.00 13.79 C ATOM 311 C LEU A 30 22.606 1.398 57.639 1.00 15.47 C ATOM 312 O LEU A 30 22.095 2.178 58.420 1.00 19.16 O ATOM 314 N LYS A 31 21.916 0.608 56.880 1.00 16.56 N ATOM 315 CA LYS A 31 20.435 0.542 57.010 1.00 16.90 C ATOM 317 CB LYS A 31 19.967 0.052 58.412 1.00 17.83 C ATOM 324 C LYS A 31 19.707 1.845 56.679 1.00 16.92 C ATOM 325 O LYS A 31 18.606 2.161 57.233 1.00 18.10 O ATOM 327 N TRP A 32 20.280 2.654 55.794 1.00 14.03 N ATOM 328 CA TRP A 32 19.619 3.894 55.463 1.00 12.43 C ATOM 330 CB TRP A 32 20.553 4.837 54.691 1.00 11.17 C ATOM 333 CG TRP A 32 21.612 5.473 55.539 1.00 10.85 C ATOM 334 CD1 TRP A 32 21.845 5.269 56.865 1.00 11.79 C ATOM 336 NE1 TRP A 32 22.923 6.044 57.280 1.00 12.05 N ATOM 338 CE2 TRP A 32 23.415 6.724 56.214 1.00 11.63 C ATOM 339 CD2 TRP A 32 22.618 6.389 55.092 1.00 10.21 C ATOM 340 CE3 TRP A 32 22.904 6.982 53.869 1.00 10.29 C ATOM 342 CZ3 TRP A 32 23.969 7.858 53.782 1.00 12.53 C ATOM 344 CH2 TRP A 32 24.726 8.154 54.907 1.00 11.69 C ATOM 346 CZ2 TRP A 32 24.448 7.626 56.126 1.00 10.84 C ATOM 348 C TRP A 32 18.371 3.523 54.650 1.00 11.50 C ATOM 349 O TRP A 32 18.311 2.522 53.944 1.00 12.52 O ATOM 351 N VAL A 33 17.375 4.377 54.719 1.00 11.31 N ATOM 352 CA VAL A 33 16.065 4.099 54.102 1.00 10.50 C ATOM 354 CB VAL A 33 14.960 4.610 55.034 1.00 10.68 C ATOM 356 CG1 VAL A 33 13.594 4.610 54.343 1.00 10.00 C ATOM 360 CG2 VAL A 33 14.951 3.783 56.320 1.00 11.80 C ATOM 364 C VAL A 33 16.043 4.779 52.728 1.00 10.58 C ATOM 365 O VAL A 33 16.485 5.929 52.557 1.00 11.61 O ATOM 367 N THR A 34 15.540 4.048 51.738 1.00 9.11 N ATOM 368 CA THR A 34 15.419 4.579 50.386 1.00 9.33 C ATOM 370 CB THR A 34 16.195 3.711 49.392 1.00 10.58 C ATOM 372 OG1 THR A 34 15.703 2.367 49.443 1.00 12.23 O ATOM 374 CG2 THR A 34 17.684 3.722 49.746 1.00 11.70 C ATOM 378 C THR A 34 13.937 4.638 49.958 1.00 9.28 C ATOM 379 O THR A 34 13.072 3.984 50.540 1.00 10.10 O ATOM 381 N PHE A 35 13.676 5.457 48.934 1.00 8.75 N ATOM 382 CA PHE A 35 12.338 5.626 48.370 1.00 8.92 C ATOM 384 CB PHE A 35 11.574 6.664 49.204 1.00 9.31 C ATOM 387 CG PHE A 35 10.237 7.021 48.650 1.00 10.11 C ATOM 388 CD1 PHE A 35 9.107 6.312 49.017 1.00 10.65 C ATOM 390 CE1 PHE A 35 7.868 6.655 48.510 1.00 12.03 C ATOM 392 CZ PHE A 35 7.761 7.700 47.584 1.00 10.81 C ATOM 394 CE2 PHE A 35 8.859 8.421 47.249 1.00 11.68 C ATOM 396 CD2 PHE A 35 10.098 8.088 47.764 1.00 10.45 C ATOM 398 C PHE A 35 12.483 6.122 46.940 1.00 9.83 C ATOM 399 O PHE A 35 13.330 6.968 46.666 1.00 10.00 O ATOM 401 N PRO A 36 11.600 5.685 46.025 1.00 9.75 N ATOM 402 CA PRO A 36 10.481 4.741 46.162 1.00 10.89 C ATOM 404 CB PRO A 36 9.557 5.200 45.048 1.00 10.66 C ATOM 407 CG PRO A 36 10.437 5.623 44.008 1.00 10.68 C ATOM 410 CD PRO A 36 11.597 6.320 44.697 1.00 10.68 C ATOM 413 C PRO A 36 10.812 3.244 45.986 1.00 10.85 C ATOM 414 O PRO A 36 9.860 2.383 45.907 1.00 12.56 O ATOM 415 N GLN A 37 12.093 2.887 45.961 1.00 11.74 N ATOM 416 CA GLN A 37 12.490 1.460 45.869 1.00 12.44 C ATOM 418 CB GLN A 37 12.088 0.679 47.099 1.00 12.50 C ATOM 421 CG GLN A 37 12.730 1.215 48.358 1.00 14.22 C ATOM 424 CD GLN A 37 12.498 0.370 49.582 1.00 16.16 C ATOM 425 OE1 GLN A 37 11.862 −0.674 49.520 1.00 21.49 O ATOM 426 NE2 GLN A 37 12.953 0.843 50.710 1.00 22.36 N ATOM 429 C GLN A 37 12.015 0.793 44.586 1.00 11.98 C ATOM 430 O GLN A 37 11.424 −0.304 44.594 1.00 15.13 O ATOM 432 N VAL A 38 12.253 1.474 43.488 1.00 12.06 N ATOM 433 CA VAL A 38 11.939 0.961 42.159 1.00 12.33 C ATOM 435 CB VAL A 38 11.084 1.982 41.362 1.00 12.47 C ATOM 437 CG1 VAL A 38 9.796 2.290 42.117 1.00 13.77 C ATOM 441 CG2 VAL A 38 11.830 3.262 41.037 1.00 13.76 C ATOM 445 C VAL A 38 13.248 0.578 41.452 1.00 13.01 C ATOM 446 O VAL A 38 14.332 0.723 42.011 1.00 12.39 O ATOM 448 N ASP A 39 13.131 0.043 40.242 1.00 13.10 N ATOM 449 CA ASP A 39 14.318 −0.237 39.441 1.00 14.56 C ATOM 451 CB ASP A 39 13.911 −0.652 38.020 1.00 15.71 C ATOM 454 CG ASP A 39 13.239 −2.028 37.949 1.00 20.14 C ATOM 455 OD1 ASP A 39 13.261 −2.810 38.933 1.00 27.15 O ATOM 456 OD2 ASP A 39 12.740 −2.346 36.852 1.00 25.39 O ATOM 457 C ASP A 39 15.199 0.996 39.390 1.00 12.97 C ATOM 458 O ASP A 39 14.705 2.129 39.202 1.00 13.90 O ATOM 460 N GLY A 40 16.503 0.789 39.588 1.00 12.14 N ATOM 461 CA GLY A 40 17.447 1.873 39.553 1.00 11.59 C ATOM 464 C GLY A 40 17.750 2.479 40.907 1.00 10.89 C ATOM 465 O GLY A 40 18.521 3.419 41.007 1.00 11.12 O ATOM 467 N GLN A 41 17.097 1.967 41.946 1.00 11.44 N ATOM 468 CA GLN A 41 17.245 2.470 43.296 1.00 10.98 C ATOM 470 CB GLN A 41 16.397 1.644 44.282 1.00 12.13 C ATOM 473 CG GLN A 41 16.743 0.139 44.297 1.00 12.95 C ATOM 476 CD GLN A 41 15.707 −0.648 45.075 1.00 15.14 C ATOM 477 OE1 GLN A 41 15.550 −0.435 46.256 1.00 18.59 O ATOM 478 NE2 GLN A 41 15.003 −1.543 44.411 1.00 22.18 N ATOM 481 C GLN A 41 18.683 2.545 43.803 1.00 10.18 C ATOM 482 O GLN A 41 19.559 1.787 43.358 1.00 10.73 O ATOM 484 N TRP A 42 18.913 3.449 44.758 1.00 9.29 N ATOM 485 CA TRP A 42 20.175 3.475 45.500 1.00 8.62 C ATOM 487 CB TRP A 42 20.067 4.399 46.695 1.00 9.35 C ATOM 490 CG TRP A 42 20.068 5.871 46.455 1.00 8.24 C ATOM 491 CD1 TRP A 42 18.985 6.677 46.392 1.00 9.84 C ATOM 493 NE1 TRP A 42 19.362 7.987 46.236 1.00 9.90 N ATOM 495 CE2 TRP A 42 20.736 8.048 46.247 1.00 8.26 C ATOM 496 CD2 TRP A 42 21.209 6.727 46.362 1.00 8.34 C ATOM 497 CE3 TRP A 42 22.600 6.506 46.347 1.00 9.51 C ATOM 499 CZ3 TRP A 42 23.436 7.588 46.260 1.00 9.12 C ATOM 501 CH2 TRP A 42 22.956 8.883 46.090 1.00 11.10 C ATOM 503 CZ2 TRP A 42 21.601 9.144 46.088 1.00 9.47 C ATOM 505 C TRP A 42 20.501 2.061 46.079 1.00 9.65 C ATOM 506 O TRP A 42 16.585 1.348 46.537 1.00 11.45 O ATOM 508 N GLU A 43 21.772 1.681 46.012 1.00 9.40 N ATOM 509 CA GLU A 43 22.284 0.387 46.512 1.00 10.45 C ATOM 511 CB AGLU A 43 22.746 −0.490 45.364 0.50 10.06 C ATOM 512 CB BGLU A 43 22.784 −0.537 45.338 0.50 10.16 C ATOM 517 CG AGLU A 43 21.634 −0.910 44.427 0.50 12.10 C ATOM 518 CG BGLU A 43 23.036 −2.043 45.750 0.50 10.29 C ATOM 523 CD AGLU A 43 22.142 −1.894 43.380 0.50 14.51 C ATOM 524 CD BGLU A 43 23.688 −3.052 44.695 0.50 13.19 C ATOM 525 OE1 AGLU A 43 23.302 −2.357 43.490 0.50 20.44 O ATOM 526 OE1 BGLU A 43 23.946 −2.788 43.499 0.50 12.08 O ATOM 527 OE2 AGLU A 43 21.367 −2.226 42.455 0.50 22.84 O ATOM 528 OE2 BGLU A 43 23.962 −4.202 45.106 0.50 15.03 O ATOM 529 C GLU A 43 23.411 0.602 47.474 1.00 9.78 C ATOM 530 O GLU A 43 24.319 1.419 47.216 1.00 9.79 O ATOM 532 N GLU A 44 23.423 −0.158 48.567 1.00 8.15 N ATOM 533 CA GLU A 44 24.572 −0.124 49.511 1.00 9.00 C ATOM 535 CB GLU A 44 24.095 −0.475 50.912 1.00 10.13 C ATOM 538 CG GLU A 44 25.149 −0.187 51.988 1.00 10.06 C ATOM 541 CD GLU A 44 24.692 −0.489 53.391 1.00 11.49 C ATOM 542 OE1 GLU A 44 23.500 −0.851 53.554 1.00 12.88 O ATOM 543 OE2 GLU A 44 25.529 −0.343 54.332 1.00 12.69 O ATOM 544 C GLU A 44 25.636 −1.122 49.065 1.00 9.85 C ATOM 545 O GLU A 44 25.290 −2.321 48.823 1.00 10.25 O ATOM 547 N LEU A 45 26.879 −0.659 48.967 1.00 10.70 N ATOM 548 CA LEU A 45 28.020 −1.454 48.498 1.00 12.02 C ATOM 550 CB LEU A 45 28.459 −0.992 47.102 1.00 12.79 C ATOM 553 CG LEU A 45 27.360 −1.452 46.135 1.00 15.99 C ATOM 555 CD1 LEU A 45 26.815 −0.440 45.202 1.00 20.04 C ATOM 559 CD2 LEU A 45 27.663 −2.822 45.541 1.00 15.67 C ATOM 563 C LEU A 45 29.217 −1.302 49.411 1.00 12.37 C ATOM 564 O LEU A 45 29.462 −0.213 49.957 1.00 12.81 O ATOM 566 N ASER A 46 29.991 −2.366 49.589 0.50 13.04 N ATOM 567 N BSER A 46 29.981 −2.386 49.551 0.50 12.82 N ATOM 568 CA ASER A 46 31.244 −2.192 50.328 0.50 13.68 C ATOM 569 CA BSER A 46 31.300 −2.357 50.215 0.50 13.35 C ATOM 572 CB ASER A 46 31.713 −3.484 50.957 0.50 14.34 C ATOM 573 CB BSER A 46 31.770 −3.818 50.405 0.50 13.78 C ATOM 578 OG ASER A 46 32.852 −3.193 51.749 0.50 17.56 O ATOM 579 OG BSER A 46 32.829 −3.971 51.338 0.50 16.91 O ATOM 582 C ASER A 46 32.362 −1.625 49.451 0.50 13.16 C ATOM 583 C BSER A 46 32.319 −1.559 49.362 0.50 12.99 C ATOM 584 O ASER A 46 32.724 −2.219 48.426 0.50 14.44 O ATOM 585 O BSER A 46 32.524 −1.885 48.203 0.50 14.64 O ATOM 588 N GLY A 47 32.931 −0.516 49.911 1.00 12.98 N ATOM 589 CA GLY A 47 34.067 0.174 49.264 1.00 12.65 C ATOM 592 C GLY A 47 35.295 0.132 50.139 1.00 12.62 C ATOM 593 O GLY A 47 35.322 −0.585 51.123 1.00 11.81 O ATOM 595 N LEU A 48 36.324 0.859 49.719 1.00 12.18 N ATOM 596 CA LEU A 48 37.609 0.936 50.440 1.00 13.20 C ATOM 598 CB LEU A 48 38.703 0.319 49.567 1.00 14.06 C ATOM 601 CG LEU A 48 38.539 −1.139 49.254 1.00 16.14 C ATOM 603 CD1 LEU A 48 39.564 −1.533 48.222 1.00 17.56 C ATOM 607 CD2 LEU A 48 38.722 −1.970 50.503 1.00 15.62 C ATOM 611 C LEU A 48 37.989 2.347 50.759 1.00 14.96 C ATOM 612 O LEU A 48 37.853 3.224 49.886 1.00 17.67 O ATOM 614 N ASP A 49 38.457 2.587 51.982 1.00 14.25 N ATOM 615 CA ASP A 49 38.898 3.925 52.362 1.00 15.81 C ATOM 617 CB ASP A 49 38.508 4.250 53.792 1.00 15.75 C ATOM 620 CG ASP A 49 39.232 3.414 54.842 1.00 16.73 C ATOM 621 OD1 ASP A 49 40.250 2.750 54.552 1.00 16.95 O ATOM 622 OD2 ASP A 49 38.742 3.421 56.007 1.00 23.27 O ATOM 623 C ASP A 49 40.407 4.086 52.098 1.00 15.71 C ATOM 624 O ASP A 49 40.991 3.232 51.452 1.00 15.44 O ATOM 626 N GLU A 50 40.984 5.201 52.574 1.00 16.80 N ATOM 627 CA GLU A 50 42.447 5.505 52.380 1.00 16.83 C ATOM 629 CB GLU A 50 42.754 6.946 52.825 1.00 16.31 C ATOM 632 CG GLU A 50 42.096 7.950 52.002 1.00 17.42 C ATOM 635 CD GLU A 50 42.520 9.388 52.332 1.00 15.16 C ATOM 636 OE1 GLU A 50 43.455 9.619 53.192 1.00 14.33 O ATOM 637 OE2 GLU A 50 41.849 10.255 51.735 1.00 15.53 O ATOM 638 C GLU A 50 43.427 4.558 53.046 1.00 19.00 C ATOM 639 O GLU A 50 44.650 4.553 52.703 1.00 19.95 O ATOM 641 N GLU A 51 42.934 3.766 53.982 1.00 19.32 N ATOM 642 CA GLU A 51 43.741 2.769 54.672 1.00 20.38 C ATOM 644 CB GLU A 51 43.417 2.808 56.159 1.00 21.41 C ATOM 647 CG GLU A 51 43.711 4.160 56.784 1.00 23.95 C ATOM 650 CD GLU A 51 43.500 4.152 58.257 1.00 24.68 C ATOM 651 OE1 GLU A 51 44.373 3.593 58.950 1.00 30.31 O ATOM 652 OE2 GLU A 51 42.488 4.733 58.706 1.00 32.21 O ATOM 653 C GLU A 51 43.466 1.384 54.124 1.00 19.92 C ATOM 654 O GLU A 51 43.931 0.378 54.697 1.00 19.56 O ATOM 656 N GLN A 52 42.717 1.327 53.020 1.00 18.89 N ATOM 657 CA GLN A 52 42.328 0.054 52.400 1.00 18.69 C ATOM 659 CB GLN A 52 43.549 −0.779 52.000 1.00 19.90 C ATOM 666 C GLN A 52 41.423 −0.789 53.285 1.00 18.80 C ATOM 667 O GLN A 52 41.434 −2.040 53.172 1.00 19.32 O ATOM 669 N HIS A 53 40.663 −0.138 54.166 1.00 17.19 N ATOM 670 CA HIS A 53 39.715 −0.828 55.003 1.00 15.92 C ATOM 672 CB HIS A 53 39.600 −0.162 56.376 1.00 17.51 C ATOM 675 CG HIS A 53 40.864 −0.204 57.199 1.00 20.93 C ATOM 676 ND1 HIS A 53 41.949 −0.994 56.884 1.00 27.78 N ATOM 678 CE1 HIS A 53 42.906 −0.804 57.780 1.00 26.65 C ATOM 680 NE2 HIS A 53 42.474 0.073 58.669 1.00 27.47 N ATOM 682 CD2 HIS A 53 41.199 0.457 58.334 1.00 27.24 C ATOM 684 C HIS A 53 38.357 −0.770 54.326 1.00 14.19 C ATOM 685 O HIS A 53 38.030 0.211 53.651 1.00 13.34 O ATOM 687 N SER A 54 37.560 −1.810 54.539 1.00 13.06 N ATOM 688 CA ASER A 54 36.206 −1.870 53.974 0.50 12.48 C ATOM 689 CA BSER A 54 36.210 −1.865 53.965 0.50 12.34 C ATOM 692 CB ASER A 54 35.624 −3.262 54.156 0.50 12.71 C ATOM 693 CB BSER A 54 35.628 −3.265 54.097 0.50 12.50 C ATOM 698 OG ASER A 54 34.435 −3.423 53.403 0.50 14.69 O ATOM 699 OG BSER A 54 36.326 −4.201 53.280 0.50 13.56 O ATOM 702 C SER A 54 35.297 −0.859 54.655 1.00 12.10 C ATOM 703 O SER A 54 35.269 −0.787 55.896 1.00 13.90 O ATOM 705 N VAL A 55 34.535 −0.100 53.854 1.00 11.09 N ATOM 706 CA VAL A 55 33.566 0.891 54.371 1.00 10.54 C ATOM 708 CB VAL A 55 34.114 2.330 54.277 1.00 10.19 C ATOM 710 CG1 VAL A 55 35.310 2.461 55.167 1.00 13.09 C ATOM 714 CG2 VAL A 55 34.460 2.665 52.856 1.00 11.17 C ATOM 718 C VAL A 55 32.238 0.771 53.602 1.00 10.18 C ATOM 719 O VAL A 55 32.214 0.333 52.462 1.00 11.24 O ATOM 721 N ARG A 56 31.135 1.140 54.233 1.00 9.01 N ATOM 722 CA ARG A 56 29.826 1.113 53.595 1.00 9.91 C ATOM 724 CB ARG A 56 28.716 1.048 54.615 1.00 10.97 C ATOM 727 CG ARG A 56 28.801 −0.147 55.563 1.00 11.58 C ATOM 730 CD ARG A 56 29.104 −1.447 54.865 1.00 15.21 C ATOM 733 NE ARG A 56 28.040 −1.854 53.969 1.00 17.47 N ATOM 735 CZ ARG A 56 28.133 −2.887 53.129 1.00 20.77 C ATOM 736 NH1 ARG A 56 27.092 −3.224 52.358 1.00 22.08 N ATOM 739 NH2 ARG A 56 29.247 −3.592 53.071 1.00 21.45 N ATOM 742 C ARG A 56 29.663 2.399 52.762 1.00 8.97 C ATOM 743 O ARG A 56 29.967 3.514 53.222 1.00 9.94 O ATOM 745 N THR A 57 29.169 2.213 51.535 1.00 9.12 N ATOM 746 CA THR A 57 28.921 3.316 50.591 1.00 8.61 C ATOM 748 CB THR A 57 29.972 3.361 49.481 1.00 9.01 C ATOM 750 OG1 THR A 57 29.798 2.254 48.563 1.00 9.10 O ATOM 752 CG2 THR A 57 31.412 3.330 50.074 1.00 9.29 C ATOM 756 C THR A 57 27.549 3.129 49.941 1.00 9.15 C ATOM 757 O THR A 57 26.953 2.050 50.011 1.00 8.65 O ATOM 759 N TYR A 58 27.067 4.170 49.276 1.00 8.82 N ATOM 760 CA TYR A 58 25.812 4.073 48.489 1.00 9.14 C ATOM 762 CB TYR A 58 24.694 4.925 49.121 1.00 10.04 C ATOM 765 CG TYR A 58 24.006 4.219 50.253 1.00 9.55 C ATOM 766 CD1 TYR A 58 23.002 3.253 50.008 1.00 9.24 C ATOM 768 CE1 TYR A 58 22.357 2.622 51.048 1.00 9.04 C ATOM 770 CZ TYR A 58 22.793 2.824 52.344 1.00 10.33 C ATOM 771 OH TYR A 58 22.181 2.115 53.417 1.00 13.23 O ATOM 773 CE2 TYR A 58 23.759 3.767 52.620 1.00 9.75 C ATOM 775 CD2 TYR A 58 24.382 4.456 51.562 1.00 10.04 C ATOM 777 C TYR A 58 26.066 4.544 47.058 1.00 9.09 C ATOM 778 O TYR A 58 26.751 5.552 46.857 1.00 9.85 O ATOM 780 N GLU A 59 25.509 3.823 46.085 1.00 7.72 N ATOM 781 CA GLU A 59 25.622 4.186 44.660 1.00 8.04 C ATOM 783 CB GLU A 59 26.491 3.177 43.912 1.00 9.06 C ATOM 786 CG GLU A 59 27.910 3.032 44.412 1.00 8.60 C ATOM 789 CD GLU A 59 28.738 2.070 43.535 1.00 9.81 C ATOM 790 OE1 GLU A 59 28.170 1.590 42.538 1.00 9.82 O ATOM 791 OE2 GLU A 59 29.925 1.884 43.849 1.00 12.02 O ATOM 792 C GLU A 59 24.248 4.212 44.000 1.00 9.20 C ATOM 793 O GLU A 59 23.337 3.488 44.368 1.00 8.68 O ATOM 795 N VAL A 60 24.140 5.064 42.988 1.00 8.44 N ATOM 796 CA VAL A 60 22.956 5.146 42.127 1.00 7.85 C ATOM 798 CB VAL A 60 21.914 6.133 42.665 1.00 8.42 C ATOM 800 CG1 VAL A 60 22.382 7.596 42.623 1.00 8.14 C ATOM 804 CG2 VAL A 60 20.563 5.969 41.977 1.00 10.07 C ATOM 808 C VAL A 60 23.429 5.545 40.724 1.00 8.37 C ATOM 809 O VAL A 60 24.353 6.371 40.582 1.00 8.58 O ATOM 811 N CYS A 61 22.861 4.928 39.697 1.00 8.88 N ATOM 812 CA CYS A 61 23.260 5.245 38.330 1.00 9.45 C ATOM 814 CB CYS A 61 24.553 4.536 37.981 1.00 9.31 C ATOM 817 SG CYS A 61 25.313 5.075 36.382 1.00 9.25 S ATOM 819 C CYS A 61 22.136 4.912 37.310 1.00 10.12 C ATOM 820 O CYS A 61 22.342 4.133 36.386 1.00 10.79 O ATOM 822 N ASP A 62 20.988 5.558 37.497 1.00 9.85 N ATOM 823 CA ASP A 62 19.793 5.376 36.660 1.00 9.28 C ATOM 825 CB ASP A 62 18.546 5.341 37.553 1.00 10.45 C ATOM 828 CG ASP A 62 17.292 5.094 36.806 1.00 10.86 C ATOM 829 OD1 ASP A 62 17.381 4.800 35.577 1.00 12.22 O ATOM 830 OD2 ASP A 62 16.194 5.144 37.448 1.00 11.23 O ATOM 831 C ASP A 62 19.788 6.503 35.646 1.00 11.31 C ATOM 832 O ASP A 62 19.204 7.568 35.876 1.00 12.18 O ATOM 834 N VAL A 63 20.458 6.261 34.514 1.00 11.89 N ATOM 835 CA VAL A 63 20.638 7.279 33.478 1.00 13.16 C ATOM 837 CB VAL A 63 22.122 7.642 33.304 1.00 13.22 C ATOM 839 CG1 VAL A 63 22.649 8.312 34.536 1.00 12.96 C ATOM 843 CG2 VAL A 63 22.963 6.406 32.886 1.00 14.83 C ATOM 847 C VAL A 63 20.081 6.837 32.138 1.00 14.23 C ATOM 848 O VAL A 63 19.822 7.649 31.266 1.00 16.46 O ATOM 850 N GLN A 64 19.887 5.542 31.985 1.00 15.84 N ATOM 851 CA GLN A 64 19.426 4.983 30.720 1.00 18.13 C ATOM 853 CB GLN A 64 20.275 3.754 30.377 1.00 18.23 C ATOM 860 C GLN A 64 17.948 4.668 30.812 1.00 19.94 C ATOM 861 O GLN A 64 17.161 5.116 29.956 1.00 22.90 O ATOM 863 N ARG A 65 17.549 3.933 31.849 1.00 20.14 N ATOM 864 CA ARG A 65 16.182 3.488 32.024 1.00 19.89 C ATOM 866 CB ARG A 65 16.116 2.460 33.151 1.00 21.74 C ATOM 875 C ARG A 65 15.242 4.657 32.342 1.00 19.41 C ATOM 876 O ARG A 65 14.266 4.904 31.598 1.00 18.18 O ATOM 878 N ALA A 66 15.523 5.374 33.434 1.00 17.78 N ATOM 879 CA ALA A 66 14.647 6.477 33.834 1.00 15.90 C ATOM 881 CB ALA A 66 13.568 6.005 34.787 1.00 17.23 C ATOM 885 C ALA A 66 15.425 7.631 34.441 1.00 14.82 C ATOM 886 O ALA A 66 15.431 7.807 35.652 1.00 12.98 O ATOM 888 N PRO A 67 16.086 8.439 33.602 1.00 13.82 N ATOM 889 CA PRO A 67 16.815 9.560 34.155 1.00 14.16 C ATOM 891 CB PRO A 67 17.626 10.096 32.966 1.00 13.92 C ATOM 894 CG PRO A 67 16.887 9.608 31.762 1.00 13.64 C ATOM 897 CD PRO A 67 16.213 8.342 32.130 1.00 13.56 C ATOM 900 C PRO A 67 15.933 10.643 34.787 1.00 13.32 C ATOM 901 O PRO A 67 16.461 11.477 35.529 1.00 14.01 O ATOM 902 N GLY A 68 14.608 10.628 34.558 1.00 12.54 N ATOM 903 CA GLY A 68 13.755 11.553 35.252 1.00 12.21 C ATOM 906 C GLY A 68 13.158 10.971 36.523 1.00 11.44 C ATOM 907 O GLY A 68 12.387 11.656 37.202 1.00 12.78 O ATOM 909 N GLN A 69 13.483 9.724 36.869 1.00 10.14 N ATOM 910 CA GLN A 69 12.998 9.159 38.153 1.00 10.41 C ATOM 912 CB GLN A 69 13.012 7.621 38.111 1.00 10.00 C ATOM 915 CG GLN A 69 12.675 6.939 39.427 1.00 9.92 C ATOM 918 CD GLN A 69 11.232 7.122 39.827 1.00 10.80 C ATOM 919 OE1 GLN A 69 10.307 6.749 39.064 1.00 13.37 O ATOM 920 NE2 GLN A 69 11.030 7.676 41.007 1.00 9.15 N ATOM 923 C GLN A 69 13.846 9.645 39.331 1.00 10.33 C ATOM 924 O GLN A 69 15.071 9.470 39.355 1.00 9.39 O ATOM 926 N ALA A 70 13.228 10.239 40.332 1.00 9.62 N ATOM 927 CA ALA A 70 13.970 10.585 41.559 1.00 9.05 C ATOM 929 CB ALA A 70 13.269 11.657 42.362 1.00 10.32 C ATOM 933 C ALA A 70 14.159 9.350 42.419 1.00 9.58 C ATOM 934 O ALA A 70 13.252 8.543 42.597 1.00 11.06 O ATOM 936 N HIS A 71 15.345 9.286 43.028 1.00 7.99 N ATOM 937 CA HIS A 71 15.731 8.234 43.969 1.00 9.09 C ATOM 939 CB HIS A 71 16.817 7.328 43.364 1.00 9.38 C ATOM 942 CG HIS A 71 16.411 6.620 42.113 1.00 9.04 C ATOM 943 ND1 HIS A 71 15.569 5.527 42.122 1.00 10.50 N ATOM 945 CE1 HIS A 71 15.444 5.073 40.884 1.00 9.85 C ATOM 947 NE2 HIS A 71 16.158 5.834 40.073 1.00 8.23 N ATOM 949 CD2 HIS A 71 16.779 6.806 40.817 1.00 10.81 C ATOM 951 C HIS A 71 16.249 8.919 45.224 1.00 8.98 C ATOM 952 O HIS A 71 17.191 9.718 45.170 1.00 9.27 O ATOM 954 N TRP A 72 15.621 8.599 46.357 1.00 8.10 N ATOM 955 CA TRP A 72 15.913 9.199 47.655 1.00 8.30 C ATOM 957 CB TRP A 72 14.601 9.697 48.329 1.00 8.49 C ATOM 960 CG TRP A 72 13.929 10.870 47.685 1.00 8.08 C ATOM 961 CD1 TRP A 72 13.113 10.859 46.603 1.00 9.35 C ATOM 963 NE1 TRP A 72 12.671 12.153 46.316 1.00 10.16 N ATOM 965 CE2 TRP A 72 13.200 13.002 47.244 1.00 10.46 C ATOM 966 CD2 TRP A 72 14.006 12.234 48.118 1.00 8.67 C ATOM 967 CE3 TRP A 72 14.663 12.862 49.162 1.00 10.76 C ATOM 969 CZ3 TRP A 72 14.533 14.248 49.299 1.00 9.32 C ATOM 971 CH2 TRP A 72 13.704 14.953 48.451 1.00 8.41 C ATOM 973 CZ2 TRP A 72 13.057 14.361 47.395 1.00 10.47 C ATOM 975 C TRP A 72 16.587 8.259 48.621 1.00 8.56 C ATOM 976 O TRP A 72 16.277 7.069 48.685 1.00 8.28 O ATOM 978 N LEU A 73 17.454 8.841 49.457 1.00 8.33 N ATOM 979 CA LEU A 73 18.276 8.127 50.455 1.00 8.49 C ATOM 981 CB LEU A 73 19.702 8.013 49.922 1.00 8.82 C ATOM 984 CG LEU A 73 20.739 7.348 50.793 1.00 8.48 C ATOM 986 CD1 LEU A 73 20.455 5.895 51.007 1.00 9.79 C ATOM 990 CD2 LEU A 73 22.131 7.516 50.167 1.00 9.77 C ATOM 994 C LEU A 73 18.274 8.950 51.732 1.00 8.70 C ATOM 995 O LEU A 73 18.618 10.119 51.712 1.00 9.55 O ATOM 997 N ARG A 74 17.906 8.347 52.844 1.00 8.25 N ATOM 998 CA ARG A 74 17.804 9.028 54.143 1.00 8.92 C ATOM 1000 CB ARG A 74 16.337 9.019 54.599 1.00 8.19 C ATOM 1003 CG ARG A 74 16.044 9.851 55.867 1.00 9.06 C ATOM 1006 CD ARG A 74 14.587 9.693 56.300 1.00 8.70 C ATOM 1009 NE ARG A 74 14.322 8.348 56.834 1.00 9.15 N ATOM 1011 CZ ARG A 74 13.146 7.711 56.777 1.00 10.02 C ATOM 1012 NH1 ARG A 74 12.107 8.206 56.136 1.00 8.19 N ATOM 1015 NH2 ARG A 74 13.043 6.513 57.364 1.00 10.25 N ATOM 1018 C ARG A 74 18.640 8.368 55.231 1.00 8.94 C ATOM 1019 O ARG A 74 18.580 7.140 55.431 1.00 8.17 O ATOM 1021 N THR A 75 19.362 9.187 55.991 1.00 8.60 N ATOM 1022 CA THR A 75 20.146 8.679 57.112 1.00 8.62 C ATOM 1024 CB THR A 75 21.096 9.767 57.658 1.00 8.78 C ATOM 1026 OG1 THR A 75 20.367 10.829 58.287 1.00 8.90 O ATOM 1028 CG2 THR A 75 21.959 10.364 56.585 1.00 10.28 C ATOM 1032 C THR A 75 19.244 8.253 58.264 1.00 8.43 C ATOM 1033 O THR A 75 18.035 8.532 58.278 1.00 8.19 O ATOM 1035 N GLY A 76 19.817 7.593 59.259 1.00 9.46 N ATOM 1036 CA GLY A 76 19.152 7.453 60.520 1.00 8.62 C ATOM 1039 C GLY A 76 18.957 8.781 61.212 1.00 8.90 C ATOM 1040 O GLY A 76 19.496 9.813 60.773 1.00 8.59 O ATOM 1042 N TRP A 77 18.196 8.750 62.312 1.00 9.17 N ATOM 1043 CA TRP A 77 17.969 9.943 63.121 1.00 8.14 C ATOM 1045 CB TRP A 77 16.779 9.726 64.058 1.00 8.44 C ATOM 1048 CG TRP A 77 16.293 10.907 64.819 1.00 7.96 C ATOM 1049 CD1 TRP A 77 15.678 12.024 64.318 1.00 9.29 C ATOM 1051 NE1 TRP A 77 15.356 12.889 65.323 1.00 8.84 N ATOM 1053 CE2 TRP A 77 15.705 12.319 66.525 1.00 11.12 C ATOM 1054 CD2 TRP A 77 16.302 11.073 66.245 1.00 9.62 C ATOM 1055 CE3 TRP A 77 16.786 10.300 67.306 1.00 10.69 C ATOM 1057 CZ3 TRP A 77 16.600 10.783 68.622 1.00 10.71 C ATOM 1059 CH2 TRP A 77 15.994 12.028 68.843 1.00 9.85 C ATOM 1061 CZ2 TRP A 77 15.526 12.791 67.825 1.00 10.22 C ATOM 1063 C TRP A 77 19.232 10.253 63.938 1.00 9.50 C ATOM 1064 O TRP A 77 19.766 9.410 64.641 1.00 10.66 O ATOM 1066 N VAL A 78 19.701 11.502 63.839 1.00 7.99 N ATOM 1067 CA VAL A 78 20.933 11.956 64.510 1.00 8.77 C ATOM 1069 CB VAL A 78 21.868 12.650 63.480 1.00 8.76 C ATOM 1071 CG1 VAL A 78 23.149 13.128 64.160 1.00 9.73 C ATOM 1075 CG2 VAL A 78 22.140 11.723 62.286 1.00 10.01 C ATOM 1079 C VAL A 78 20.588 12.924 65.636 1.00 8.96 C ATOM 1080 O VAL A 78 20.182 14.033 65.385 1.00 9.98 O ATOM 1082 N PRO A 79 20.712 12.491 66.891 1.00 10.47 N ATOM 1083 CA PRO A 79 20.522 13.409 68.016 1.00 11.43 C ATOM 1085 CB PRO A 79 20.668 12.495 69.242 1.00 12.63 C ATOM 1088 CG PRO A 79 20.418 11.101 68.726 1.00 12.60 C ATOM 1091 CD PRO A 79 20.958 11.100 67.327 1.00 12.00 C ATOM 1094 C PRO A 79 21.555 14.517 67.986 1.00 12.22 C ATOM 1095 O PRO A 79 22.738 14.224 67.784 1.00 12.62 O ATOM 1096 N ARG A 80 21.148 15.777 68.116 1.00 12.32 N ATOM 1097 CA ARG A 80 22.063 16.891 67.894 1.00 15.12 C ATOM 1099 CB ARG A 80 21.354 18.133 67.362 1.00 15.85 C ATOM 1102 CG ARG A 80 20.509 18.823 68.365 1.00 15.55 C ATOM 1105 CD ARG A 80 20.040 20.171 67.859 1.00 16.53 C ATOM 1108 NE ARG A 80 19.079 20.802 68.756 1.00 16.98 N ATOM 1110 CZ ARG A 80 19.358 21.768 69.639 1.00 19.02 C ATOM 1111 NH1 ARG A 80 20.592 22.265 69.771 1.00 18.43 N ATOM 1114 NH2 ARG A 80 18.399 22.260 70.413 1.00 20.74 N ATOM 1117 C ARG A 80 22.889 17.306 69.114 1.00 15.78 C ATOM 1118 O ARG A 80 23.793 18.125 68.960 1.00 15.13 O ATOM 1120 N ARG A 81 22.553 16.792 70.301 1.00 17.14 N ATOM 1121 CA ARG A 81 23.315 17.155 71.515 1.00 17.63 C ATOM 1123 CB ARG A 81 24.713 16.575 71.447 1.00 18.84 C ATOM 1126 CG ARG A 81 24.662 15.058 71.474 1.00 22.20 C ATOM 1129 CD ARG A 81 26.000 14.448 71.540 1.00 26.32 C ATOM 1132 NE ARG A 81 26.700 14.783 72.776 1.00 25.33 N ATOM 1134 CZ ARG A 81 28.013 14.678 72.960 1.00 28.71 C ATOM 1135 NH1 ARG A 81 28.806 14.246 71.988 1.00 30.89 N ATOM 1138 NH2 ARG A 81 28.543 15.029 74.121 1.00 30.52 N ATOM 1141 C ARG A 81 23.257 18.689 71.673 1.00 18.22 C ATOM 1142 O ARG A 81 22.206 19.255 71.469 1.00 19.54 O ATOM 1144 N GLY A 82 24.336 19.375 71.993 1.00 19.11 N ATOM 1145 CA GLY A 82 24.162 20.830 72.148 1.00 19.74 C ATOM 1148 C GLY A 82 24.141 21.627 70.843 1.00 18.66 C ATOM 1149 O GLY A 82 23.956 22.852 70.894 1.00 20.16 O ATOM 1151 N ALA A 83 24.300 20.958 69.699 1.00 16.48 N ATOM 1152 CA ALA A 83 24.749 21.647 68.486 1.00 14.41 C ATOM 1154 CB ALA A 83 25.109 20.650 67.433 1.00 14.82 C ATOM 1158 C ALA A 83 23.717 22.602 67.927 1.00 15.13 C ATOM 1159 O ALA A 83 22.590 22.179 67.717 1.00 15.30 O ATOM 1161 N VAL A 84 24.099 23.840 67.619 1.00 13.70 N ATOM 1162 CA VAL A 84 23.208 24.783 66.949 1.00 13.96 C ATOM 1164 CB VAL A 84 23.285 26.201 67.506 1.00 15.82 C ATOM 1166 CG1 VAL A 84 22.345 27.139 66.755 1.00 16.71 C ATOM 1170 CG2 VAL A 84 22.898 26.204 68.979 1.00 17.60 C ATOM 1174 C VAL A 84 23.456 24.739 65.425 1.00 11.78 C ATOM 1175 O VAL A 84 22.524 24.853 64.652 1.00 11.60 O ATOM 1177 N HIS A 85 24.702 24.618 65.002 1.00 10.16 N ATOM 1178 CA HIS A 85 25.008 24.300 63.609 1.00 10.37 C ATOM 1180 CB HIS A 85 25.807 25.405 62.924 1.00 11.74 C ATOM 1183 CG HIS A 85 25.082 26.705 62.892 1.00 15.32 C ATOM 1184 ND1 HIS A 85 24.291 27.100 61.828 1.00 18.93 N ATOM 1186 CE1 HIS A 85 23.746 28.273 62.116 1.00 13.57 C ATOM 1188 NE2 HIS A 85 24.160 28.644 63.312 1.00 18.04 N ATOM 1190 CD2 HIS A 85 24.979 27.670 63.827 1.00 15.62 C ATOM 1192 C HIS A 85 25.797 23.011 63.531 1.00 9.98 C ATOM 1193 O HIS A 85 26.639 22.773 64.417 1.00 11.68 O ATOM 1195 N VAL A 86 25.484 22.197 62.533 1.00 9.78 N ATOM 1196 CA VAL A 86 26.186 20.955 62.280 1.00 9.44 C ATOM 1198 CB VAL A 86 25.225 19.738 62.406 1.00 10.60 C ATOM 1200 CG1 VAL A 86 25.866 18.460 61.842 1.00 12.94 C ATOM 1204 CG2 VAL A 86 24.831 19.560 63.871 1.00 10.81 C ATOM 1208 C VAL A 86 26.823 21.029 60.908 1.00 10.03 C ATOM 1209 O VAL A 86 26.222 21.547 59.941 1.00 11.17 O ATOM 1211 N TYR A 87 28.043 20.519 60.790 1.00 7.91 N ATOM 1212 CA TYR A 87 28.716 20.415 59.536 1.00 7.81 C ATOM 1214 CB TYR A 87 30.196 20.779 59.709 1.00 8.49 C ATOM 1217 CG TYR A 87 30.356 22.226 60.090 1.00 8.70 C ATOM 1218 CD1 TYR A 87 30.439 23.212 59.131 1.00 10.52 C ATOM 1220 CE1 TYR A 87 30.548 24.542 59.496 1.00 11.11 C ATOM 1222 CZ TYR A 87 30.511 24.876 60.820 1.00 12.13 C ATOM 1223 OH TYR A 87 30.616 26.203 61.247 1.00 14.33 O ATOM 1225 CE2 TYR A 87 30.414 23.923 61.774 1.00 10.66 C ATOM 1227 CD2 TYR A 87 30.299 22.607 61.404 1.00 10.17 C ATOM 1229 C TYR A 87 28.605 19.024 58.963 1.00 8.80 C ATOM 1230 O TYR A 87 28.736 18.040 59.702 1.00 9.37 O ATOM 1232 N ALA A 88 28.354 18.955 57.655 1.00 7.91 N ATOM 1233 CA ALA A 88 28.258 17.688 56.937 1.00 7.02 C ATOM 1235 CB ALA A 88 26.853 17.549 56.298 1.00 7.55 C ATOM 1239 C ALA A 88 29.270 17.584 55.830 1.00 8.97 C ATOM 1240 O ALA A 88 29.123 18.283 54.818 1.00 10.24 O ATOM 1242 N THR A 89 30.290 16.765 55.996 1.00 8.71 N ATOM 1243 CA THR A 89 31.290 16.569 54.944 1.00 7.77 C ATOM 1245 CB THR A 89 32.686 16.393 55.511 1.00 8.61 C ATOM 1247 OG1 THR A 89 33.092 17.561 56.259 1.00 9.57 O ATOM 1249 CG2 THR A 89 33.675 16.151 54.393 1.00 8.17 C ATOM 1253 C THR A 89 30.899 15.346 54.149 1.00 8.06 C ATOM 1254 O THR A 89 30.762 14.265 54.682 1.00 8.00 O ATOM 1256 N LEU A 90 30.700 15.571 52.842 1.00 8.21 N ATOM 1257 CA LEU A 90 30.340 14.538 51.869 1.00 8.69 C ATOM 1259 CB LEU A 90 29.139 15.023 51.019 1.00 9.11 C ATOM 1262 CG LEU A 90 27.861 15.331 51.822 1.00 15.79 C ATOM 1264 CD1 LEU A 90 26.896 16.070 50.900 1.00 20.74 C ATOM 1268 CD2 LEU A 90 27.283 14.096 52.289 1.00 19.77 C ATOM 1272 C LEU A 90 31.508 14.291 50.920 1.00 8.92 C ATOM 1273 O LEU A 90 32.029 15.245 50.367 1.00 10.39 O ATOM 1275 N ARG A 91 31.888 13.034 50.722 1.00 8.77 N ATOM 1276 CA ARG A 91 32.911 12.662 49.732 1.00 8.68 C ATOM 1278 CB ARG A 91 34.142 12.014 50.403 1.00 9.59 C ATOM 1281 CG ARG A 91 34.773 12.963 51.396 1.00 9.66 C ATOM 1284 CD ARG A 91 36.013 12.436 52.061 1.00 11.36 C ATOM 1287 NE ARG A 91 36.469 13.307 53.142 1.00 11.87 N ATOM 1289 CZ ARG A 91 37.207 14.409 52.998 1.00 10.22 C ATOM 1290 NH1 ARG A 91 37.516 15.118 54.108 1.00 12.10 N ATOM 1293 NH2 ARG A 91 37.708 14.777 51.815 1.00 12.98 N ATOM 1296 C ARG A 91 32.241 11.759 48.729 1.00 9.26 C ATOM 1297 O ARG A 91 31.544 10.796 49.105 1.00 8.93 O ATOM 1299 N PHE A 92 32.415 12.050 47.438 1.00 7.97 N ATOM 1300 CA PHE A 92 31.661 11.373 46.418 1.00 6.88 C ATOM 1302 CB PHE A 92 30.239 12.000 46.308 1.00 7.83 C ATOM 1305 CG PHE A 92 30.221 13.431 45.837 1.00 7.27 C ATOM 1306 CD1 PHE A 92 30.056 13.745 44.471 1.00 8.11 C ATOM 1308 CE1 PHE A 92 30.042 15.066 44.056 1.00 8.18 C ATOM 1310 CZ PHE A 92 30.219 16.063 44.939 1.00 8.65 C ATOM 1312 CE2 PHE A 92 30.397 15.799 46.280 1.00 9.28 C ATOM 1314 CD2 PHE A 92 30.359 14.489 46.736 1.00 8.23 C ATOM 1316 C PHE A 92 32.347 11.364 45.069 1.00 7.47 C ATOM 1317 O PHE A 92 33.136 12.245 44.721 1.00 8.15 O ATOM 1319 N THR A 93 32.029 10.327 44.315 1.00 7.11 N ATOM 1320 CA THR A 93 32.417 10.218 42.886 1.00 7.40 C ATOM 1322 CB ATHR A 93 33.136 8.982 42.504 0.50 8.62 C ATOM 1323 CB BTHR A 93 32.705 8.657 42.619 0.50 7.27 C ATOM 1326 OG1 ATHR A 93 32.308 7.887 42.774 0.50 10.04 O ATOM 1327 OG1 BTHR A 93 33.752 8.147 43.485 0.50 7.05 O ATOM 1330 CG2 ATHR A 93 34.452 8.867 43.335 0.50 6.07 C ATOM 1331 CG2 BTHR A 93 33.038 8.353 41.171 0.50 6.94 C ATOM 1338 C THR A 93 31.212 10.559 42.044 1.00 7.68 C ATOM 1339 O THR A 93 30.073 10.205 42.401 1.00 7.10 O ATOM 1341 N MET A 94 31.442 11.283 40.954 1.00 7.62 N ATOM 1342 CA MET A 94 30.404 11.649 39.987 1.00 6.97 C ATOM 1344 CB MET A 94 30.119 13.136 40.070 1.00 7.97 C ATOM 1347 CG MET A 94 28.937 13.593 39.231 1.00 9.97 C ATOM 1350 SD MET A 94 27.301 12.983 39.741 1.00 9.34 S ATOM 1351 CE MET A 94 27.147 13.874 41.283 1.00 10.18 C ATOM 1355 C MET A 94 30.877 11.291 38.588 1.00 7.52 C ATOM 1356 O MET A 94 31.958 11.760 38.167 1.00 7.31 O ATOM 1358 N LEU A 95 30.152 10.399 37.907 1.00 7.87 N ATOM 1359 CA LEU A 95 30.511 10.025 36.542 1.00 7.80 C ATOM 1361 CB LEU A 95 29.836 8.714 36.120 1.00 7.80 C ATOM 1364 CG LEU A 95 30.586 7.423 36.437 1.00 9.86 C ATOM 1366 CD1 LEU A 95 31.820 7.207 35.574 1.00 11.31 C ATOM 1370 CD2 LEU A 95 30.907 7.368 37.943 1.00 10.48 C ATOM 1374 C LEU A 95 30.176 11.134 35.520 1.00 9.01 C ATOM 1375 O LEU A 95 29.127 11.817 35.573 1.00 9.51 O ATOM 1377 N GLU A 96 31.105 11.287 34.588 1.00 7.72 N ATOM 1378 CA GLU A 96 30.885 12.081 33.378 1.00 9.25 C ATOM 1380 CB GLU A 96 32.194 12.109 32.576 1.00 9.27 C ATOM 1383 CG GLU A 96 32.102 12.995 31.340 1.00 11.60 C ATOM 1386 CD GLU A 96 33.104 12.676 30.232 1.00 12.47 C ATOM 1387 OE1 GLU A 96 33.622 11.523 30.087 1.00 15.75 O ATOM 1388 OE2 GLU A 96 33.299 13.608 29.422 1.00 19.07 O ATOM 1389 C GLU A 96 29.757 11.441 32.528 1.00 8.51 C ATOM 1390 O GLU A 96 29.860 10.294 32.115 1.00 8.64 O ATOM 1392 N CYS A 97 28.667 12.175 32.302 1.00 9.62 N ATOM 1393 CA CYS A 97 27.535 11.669 31.529 1.00 9.42 C ATOM 1395 CB CYS A 97 26.483 12.790 31.349 1.00 8.87 C ATOM 1398 SG CYS A 97 25.542 13.065 32.851 1.00 12.10 S ATOM 1400 C CYS A 97 27.963 11.127 30.161 1.00 9.98 C ATOM 1401 O CYS A 97 27.520 10.056 29.716 1.00 9.98 O ATOM 1403 N LEU A 98 28.855 11.850 29.511 1.00 9.65 N ATOM 1404 CA LEU A 98 29.404 11.373 28.223 1.00 11.52 C ATOM 1406 CB LEU A 98 30.241 12.452 27.597 1.00 12.26 C ATOM 1409 CG LEU A 98 29.420 13.622 27.039 1.00 15.94 C ATOM 1411 CD1 LEU A 98 30.349 14.683 26.518 1.00 19.86 C ATOM 1415 CD2 LEU A 98 28.428 13.204 25.939 1.00 19.10 C ATOM 1419 C LEU A 98 30.160 10.023 28.230 1.00 10.56 C ATOM 1420 O LEU A 98 30.307 9.407 27.174 1.00 10.78 O ATOM 1422 N SER A 99 30.649 9.586 29.387 1.00 9.83 N ATOM 1423 CA SER A 99 31.226 8.259 29.520 1.00 9.91 C ATOM 1425 CB SER A 99 32.175 8.171 30.729 1.00 10.76 C ATOM 1428 OG SER A 99 33.364 8.855 30.520 1.00 11.45 O ATOM 1430 C SER A 99 30.230 7.132 29.658 1.00 11.32 C ATOM 1431 O SER A 99 30.641 5.970 29.671 1.00 12.39 O ATOM 1433 N LEU A 100 28.943 7.438 29.831 1.00 9.82 N ATOM 1434 CA LEU A 100 27.958 6.400 30.119 1.00 10.93 C ATOM 1436 CB LEU A 100 26.996 6.915 31.204 1.00 10.49 C ATOM 1439 CG LEU A 100 27.629 7.403 32.517 1.00 11.90 C ATOM 1441 CD1 LEU A 100 26.615 8.112 33.387 1.00 12.68 C ATOM 1445 CD2 LEU A 100 28.298 6.256 33.253 1.00 12.67 C ATOM 1449 C LEU A 100 27.199 6.043 28.858 1.00 10.94 C ATOM 1450 O LEU A 100 26.527 6.895 28.300 1.00 11.49 O ATOM 1452 N PRO A 101 27.328 4.802 28.376 1.00 12.49 N ATOM 1453 CA PRO A 101 26.705 4.491 27.079 1.00 14.38 C ATOM 1455 CB PRO A 101 27.164 3.049 26.780 1.00 14.56 C ATOM 1458 CG PRO A 101 27.671 2.504 28.043 1.00 13.39 C ATOM 1461 CD PRO A 101 28.072 3.664 28.932 1.00 12.86 C ATOM 1464 C PRO A 101 25.179 4.618 27.170 1.00 16.17 C ATOM 1465 O PRO A 101 24.586 4.219 28.169 1.00 17.28 O ATOM 1466 N ARG A 102 24.542 5.236 26.180 1.00 18.27 N ATOM 1467 CA ARG A 102 23.071 5.467 26.252 1.00 18.80 C ATOM 1469 CB ARG A 102 22.288 4.167 26.058 1.00 20.13 C ATOM 1478 C ARG A 102 22.501 6.186 27.484 1.00 20.78 C ATOM 1479 O ARG A 102 21.292 6.102 27.722 1.00 20.36 O ATOM 1481 N ALA A 103 23.344 6.848 28.278 1.00 21.29 N ATOM 1482 CA ALA A 103 22.902 8.075 28.944 1.00 22.02 C ATOM 1484 CB ALA A 103 24.018 8.713 29.749 1.00 21.04 C ATOM 1488 C ALA A 103 22.504 8.998 27.806 1.00 23.53 C ATOM 1489 O ALA A 103 23.080 8.919 26.705 1.00 26.11 O ATOM 1491 N GLY A 104 21.536 9.860 28.046 1.00 24.21 N ATOM 1492 CA GLY A 104 21.126 10.821 27.038 1.00 24.18 C ATOM 1495 C GLY A 104 21.141 12.226 27.582 1.00 24.08 C ATOM 1496 O GLY A 104 21.794 12.520 28.615 1.00 24.73 O ATOM 1498 N ARG A 105 20.363 13.077 26.925 1.00 23.02 N ATOM 1499 CA ARG A 105 20.319 14.505 27.210 1.00 21.51 C ATOM 1501 CB ARG A 105 19.374 15.210 26.220 1.00 22.65 C ATOM 1510 C ARG A 105 19.897 14.834 28.654 1.00 20.55 C ATOM 1511 O ARG A 105 20.291 15.867 29.153 1.00 21.64 O ATOM 1513 N SER A 106 19.133 13.946 29.300 1.00 18.47 N ATOM 1514 CA SER A 106 18.572 14.125 30.627 1.00 18.10 C ATOM 1516 CB SER A 106 17.404 13.135 30.738 1.00 17.99 C ATOM 1519 OG SER A 106 16.607 13.369 31.835 1.00 25.30 O ATOM 1521 C SER A 106 19.563 13.852 31.795 1.00 14.68 C ATOM 1522 O SER A 106 19.290 14.115 32.987 1.00 15.45 O ATOM 1524 N CYS A 107 20.708 13.282 31.443 1.00 12.87 N ATOM 1525 CA CYS A 107 21.700 12.891 32.455 1.00 11.12 C ATOM 1527 CB CYS A 107 22.817 12.123 31.753 1.00 11.59 C ATOM 1530 SG CYS A 107 24.162 11.512 32.802 1.00 12.27 S ATOM 1532 C CYS A 107 22.221 14.143 33.175 1.00 10.83 C ATOM 1533 O CYS A 107 22.444 15.198 32.548 1.00 11.33 O ATOM 1535 N LYS A 108 22.466 14.000 34.485 1.00 10.91 N ATOM 1536 CA LYS A 108 22.880 15.103 35.357 1.00 10.39 C ATOM 1538 CB LYS A 108 21.810 15.383 36.426 1.00 11.64 C ATOM 1541 CG LYS A 108 20.441 15.745 35.847 1.00 14.77 C ATOM 1544 CD LYS A 108 20.525 16.934 34.961 1.00 17.33 C ATOM 1547 CE LYS A 108 19.159 17.265 34.350 1.00 19.39 C ATOM 1550 NZ LYS A 108 19.274 18.369 33.346 1.00 25.41 N ATOM 1554 C LYS A 108 24.195 14.732 36.042 1.00 9.99 C ATOM 1555 O LYS A 108 24.544 13.535 36.099 1.00 9.47 O ATOM 1557 N GLU A 109 24.893 15.755 36.537 1.00 9.09 N ATOM 1558 CA GLU A 109 26.160 15.592 37.286 1.00 8.94 C ATOM 1560 CB GLU A 109 27.343 16.058 36.435 1.00 9.45 C ATOM 1563 CG GLU A 109 27.565 15.126 35.214 1.00 9.12 C ATOM 1566 CD GLU A 109 28.416 15.737 34.096 1.00 11.55 C ATOM 1567 OE1 GLU A 109 28.725 16.969 34.146 1.00 12.46 O ATOM 1568 OE2 GLU A 109 28.695 15.006 33.114 1.00 11.28 O ATOM 1569 C GLU A 109 26.107 16.264 38.660 1.00 9.52 C ATOM 1570 O GLU A 109 27.103 16.795 39.154 1.00 9.16 O ATOM 1572 N THR A 110 24.926 16.208 39.265 1.00 9.72 N ATOM 1573 CA THR A 110 24.634 16.848 40.560 1.00 10.44 C ATOM 1575 CB THR A 110 23.915 18.228 40.417 1.00 11.43 C ATOM 1577 OG1 THR A 110 22.567 18.038 39.910 1.00 11.72 O ATOM 1579 CG2 THR A 110 24.671 19.176 39.515 1.00 12.59 C ATOM 1583 C THR A 110 23.711 15.949 41.378 1.00 10.11 C ATOM 1584 O THR A 110 23.154 14.985 40.854 1.00 9.27 O ATOM 1586 N PHE A 111 23.621 16.248 42.666 1.00 9.89 N ATOM 1587 CA PHE A 111 22.630 15.628 43.565 1.00 9.35 C ATOM 1589 CB PHE A 111 23.124 14.385 44.258 1.00 9.32 C ATOM 1592 CG PHE A 111 24.261 14.625 45.235 1.00 9.36 C ATOM 1593 CD1 PHE A 111 24.050 14.561 46.582 1.00 11.62 C ATOM 1595 CE1 PHE A 111 25.090 14.724 47.466 1.00 11.92 C ATOM 1597 CZ PHE A 111 26.381 14.950 47.011 1.00 11.03 C ATOM 1599 CE2 PHE A 111 26.618 15.013 45.701 1.00 10.10 C ATOM 1601 CD2 PHE A 111 25.569 14.828 44.782 1.00 9.30 C ATOM 1603 C PHE A 111 22.240 16.729 44.561 1.00 10.26 C ATOM 1604 O PHE A 111 22.954 17.724 44.726 1.00 9.99 O ATOM 1606 N THR A 112 21.090 16.576 45.199 1.00 8.76 N ATOM 1607 CA THR A 112 20.624 17.601 46.133 1.00 8.93 C ATOM 1609 CB THR A 112 19.291 18.185 45.671 1.00 10.01 C ATOM 1611 OG1 THR A 112 19.472 18.700 44.324 1.00 10.82 O ATOM 1613 CG2 THR A 112 18.818 19.278 46.593 1.00 10.35 C ATOM 1617 C THR A 112 20.554 17.039 47.567 1.00 8.49 C ATOM 1618 O THR A 112 20.090 15.914 47.811 1.00 9.37 O ATOM 1620 N VAL A 113 21.025 17.864 48.492 1.00 7.91 N ATOM 1621 CA VAL A 113 21.098 17.515 49.910 1.00 8.28 C ATOM 1623 CB VAL A 113 22.472 17.809 50.495 1.00 9.20 C ATOM 1625 CG1 VAL A 113 22.472 17.446 52.027 1.00 10.87 C ATOM 1629 CG2 VAL A 113 23.518 16.997 49.742 1.00 9.25 C ATOM 1633 C VAL A 113 20.042 18.337 50.668 1.00 9.30 C ATOM 1634 O VAL A 113 19.916 19.559 50.455 1.00 8.34 O ATOM 1636 N PHE A 114 19.287 17.628 51.522 1.00 8.22 N ATOM 1637 CA PHE A 114 18.233 18.186 52.384 1.00 8.86 C ATOM 1639 CB PHE A 114 16.847 17.662 51.976 1.00 9.77 C ATOM 1642 CG PHE A 114 16.447 17.939 50.536 1.00 9.99 C ATOM 1643 CD1 PHE A 114 15.627 19.029 50.213 1.00 10.02 C ATOM 1645 CE1 PHE A 114 15.245 19.261 48.927 1.00 9.70 C ATOM 1647 CZ PHE A 114 15.702 18.393 47.902 1.00 9.70 C ATOM 1649 CE2 PHE A 114 16.454 17.308 48.214 1.00 10.86 C ATOM 1651 CD2 PHE A 114 16.851 17.081 49.512 1.00 9.83 C ATOM 1653 C PHE A 114 18.402 17.739 53.835 1.00 8.96 C ATOM 1654 O PHE A 114 19.070 16.721 54.119 1.00 8.46 O ATOM 1656 N TYR A 115 17.785 18.447 54.763 1.00 8.63 N ATOM 1657 CA TYR A 115 17.628 17.889 56.116 1.00 8.52 C ATOM 1659 CB TYR A 115 18.717 18.352 57.092 1.00 8.85 C ATOM 1662 CG TYR A 115 18.585 19.788 57.592 1.00 8.06 C ATOM 1663 CD1 TYR A 115 18.116 20.064 58.861 1.00 10.86 C ATOM 1665 CE1 TYR A 115 17.978 21.382 59.294 1.00 11.45 C ATOM 1667 CZ TYR A 115 18.312 22.416 58.461 1.00 12.69 C ATOM 1668 OH TYR A 115 18.216 23.747 58.908 1.00 15.40 O ATOM 1670 CE2 TYR A 115 18.818 22.152 57.233 1.00 12.22 C ATOM 1672 CD2 TYR A 115 18.906 20.837 56.791 1.00 8.82 C ATOM 1674 C TYR A 115 16.241 18.195 56.674 1.00 7.78 C ATOM 1675 O TYR A 115 15.548 19.067 56.162 1.00 8.01 O ATOM 1677 N TYR A 116 15.862 17.463 57.721 1.00 8.13 N ATOM 1678 CA TYR A 116 14.605 17.718 58.413 1.00 8.62 C ATOM 1680 CB TYR A 116 13.434 16.894 57.841 1.00 8.49 C ATOM 1683 CG TYR A 116 12.137 17.248 58.540 1.00 9.29 C ATOM 1684 CD1 TYR A 116 11.603 18.513 58.398 1.00 7.91 C ATOM 1686 CE1 TYR A 116 10.440 18.911 59.091 1.00 9.45 C ATOM 1688 CZ TYR A 116 9.848 18.035 59.962 1.00 11.48 C ATOM 1689 OH TYR A 116 8.746 18.469 60.719 1.00 14.30 O ATOM 1691 CE2 TYR A 116 10.383 16.747 60.125 1.00 11.31 C ATOM 1693 CD2 TYR A 116 11.542 16.394 59.459 1.00 12.12 C ATOM 1695 C TYR A 116 14.813 17.407 59.897 1.00 8.75 C ATOM 1696 O TYR A 116 15.348 16.352 60.233 1.00 8.64 O ATOM 1698 N GLU A 117 14.455 18.344 60.768 1.00 9.01 N ATOM 1699 CA GLU A 117 14.596 18.131 62.196 1.00 9.67 C ATOM 1701 CB GLU A 117 14.807 19.445 62.934 1.00 9.99 C ATOM 1704 CG GLU A 117 15.905 20.323 62.447 1.00 11.48 C ATOM 1707 CD GLU A 117 16.063 21.503 63.381 1.00 13.75 C ATOM 1708 OE1 GLU A 117 16.333 21.243 64.577 1.00 13.79 O ATOM 1709 OE2 GLU A 117 15.902 22.651 62.901 1.00 17.99 O ATOM 1710 C GLU A 117 13.331 17.504 62.789 1.00 9.86 C ATOM 1711 O GLU A 117 12.212 17.874 62.410 1.00 10.42 O ATOM 1713 N SER A 118 13.478 16.574 63.737 1.00 8.88 N ATOM 1714 CA SER A 118 12.326 15.959 64.396 1.00 9.11 C ATOM 1716 CB SER A 118 11.879 14.659 63.724 1.00 9.48 C ATOM 1719 OG SER A 118 12.881 13.657 63.864 1.00 10.18 O ATOM 1721 C SER A 118 12.681 15.716 65.875 1.00 8.87 C ATOM 1722 O SER A 118 13.854 15.513 66.202 1.00 8.37 O ATOM 1724 N ASP A 119 11.688 15.741 66.762 1.00 8.36 N ATOM 1725 CA ASP A 119 11.956 15.650 68.203 1.00 8.09 C ATOM 1727 CB ASP A 119 10.870 16.386 69.021 1.00 8.58 C ATOM 1730 CG ASP A 119 11.244 17.852 69.321 1.00 9.08 C ATOM 1731 OD1 ASP A 119 12.453 18.141 69.540 1.00 10.16 O ATOM 1732 OD2 ASP A 119 10.325 18.697 69.394 1.00 9.56 O ATOM 1733 C ASP A 119 12.159 14.204 68.678 1.00 7.79 C ATOM 1734 O ASP A 119 12.595 13.945 69.818 1.00 9.62 O ATOM 1736 N ALA A 120 11.912 13.243 67.779 1.00 7.59 N ATOM 1737 CA ALA A 120 12.149 11.813 68.070 1.00 7.40 C ATOM 1739 CB ALA A 120 10.978 11.212 68.854 1.00 8.33 C ATOM 1743 C ALA A 120 12.326 11.074 66.768 1.00 8.16 C ATOM 1744 O ALA A 120 12.084 11.616 65.707 1.00 8.25 O ATOM 1746 N ASP A 121 12.737 9.810 66.857 1.00 8.59 N ATOM 1747 CA ASP A 121 13.030 9.034 65.648 1.00 8.52 C ATOM 1749 CB ASP A 121 14.105 8.003 65.918 1.00 8.73 C ATOM 1752 CG ASP A 121 14.467 7.170 64.703 1.00 10.54 C ATOM 1753 OD1 ASP A 121 14.060 7.468 63.530 1.00 10.18 O ATOM 1754 OD2 ASP A 121 15.194 6.127 64.922 1.00 11.89 O ATOM 1755 C ASP A 121 11.746 8.379 65.182 1.00 8.94 C ATOM 1756 O ASP A 121 11.482 7.176 65.386 1.00 10.31 O ATOM 1758 N THR A 122 10.956 9.209 64.516 1.00 8.26 N ATOM 1759 CA THR A 122 9.590 8.879 64.128 1.00 8.62 C ATOM 1761 CB THR A 122 8.654 10.049 64.524 1.00 9.55 C ATOM 1763 OG1 THR A 122 9.250 11.295 64.131 1.00 10.04 O ATOM 1765 CG2 THR A 122 8.384 10.050 66.013 1.00 12.30 C ATOM 1769 C THR A 122 9.413 8.606 62.633 1.00 8.96 C ATOM 1770 O THR A 122 8.276 8.289 62.186 1.00 9.62 O ATOM 1772 N ALA A 123 10.492 8.720 61.861 1.00 8.97 N ATOM 1773 CA ALA A 123 10.395 8.461 60.406 1.00 8.00 C ATOM 1775 CB ALA A 123 11.726 8.688 59.757 1.00 7.19 C ATOM 1779 C ALA A 123 9.923 7.059 60.106 1.00 8.48 C ATOM 1780 O ALA A 123 10.148 6.108 60.915 1.00 9.30 O ATOM 1782 N THR A 124 9.256 6.923 58.946 1.00 9.17 N ATOM 1783 CA THR A 124 8.735 5.656 58.427 1.00 8.89 C ATOM 1785 CB THR A 124 7.192 5.636 58.489 1.00 9.66 C ATOM 1787 OG1 THR A 124 6.643 6.543 57.507 1.00 10.24 O ATOM 1789 CG2 THR A 124 6.696 5.997 59.853 1.00 9.70 C ATOM 1793 C THR A 124 9.171 5.492 56.967 1.00 9.28 C ATOM 1794 O THR A 124 9.957 6.280 56.448 1.00 8.87 O ATOM 1796 N ALA A 125 8.645 4.469 56.296 1.00 8.98 N ATOM 1797 CA ALA A 125 9.009 4.281 54.880 1.00 8.70 C ATOM 1799 CB ALA A 125 8.402 3.030 54.361 1.00 9.55 C ATOM 1803 C ALA A 125 8.537 5.463 54.028 1.00 8.90 C ATOM 1804 O ALA A 125 9.105 5.729 52.971 1.00 8.92 O ATOM 1806 N LEU A 126 7.476 6.146 54.487 1.00 8.75 N ATOM 1807 CA LEU A 126 6.824 7.189 53.684 1.00 8.77 C ATOM 1809 CB LEU A 126 5.325 6.855 53.591 1.00 9.83 C ATOM 1812 CG LEU A 126 4.982 5.534 52.898 1.00 12.28 C ATOM 1814 CD1 LEU A 126 3.476 5.318 52.909 1.00 13.65 C ATOM 1818 CD2 LEU A 126 5.463 5.531 51.482 1.00 13.01 C ATOM 1822 C LEU A 126 6.924 8.590 54.227 1.00 9.15 C ATOM 1823 O LEU A 126 6.493 9.552 53.551 1.00 8.84 O ATOM 1825 N THR A 127 7.436 8.732 55.448 1.00 8.64 N ATOM 1826 CA THR A 127 7.471 10.039 56.115 1.00 8.59 C ATOM 1828 CB THR A 127 6.337 10.202 57.149 1.00 9.13 C ATOM 1830 OG1 THR A 127 6.472 9.200 58.181 1.00 10.34 O ATOM 1832 CG2 THR A 127 4.938 10.113 56.529 1.00 9.97 C ATOM 1836 C THR A 127 8.798 10.216 56.857 1.00 8.79 C ATOM 1837 O THR A 127 9.374 9.219 57.290 1.00 9.36 O ATOM 1839 N PRO A 128 9.273 11.464 57.056 1.00 7.86 N ATOM 1840 CA PRO A 128 8.749 12.723 56.502 1.00 8.31 C ATOM 1842 CB PRO A 128 9.756 13.757 56.897 1.00 8.97 C ATOM 1845 CG PRO A 128 10.676 13.119 57.920 1.00 11.44 C ATOM 1848 CD PRO A 128 10.521 11.642 57.837 1.00 8.52 C ATOM 1851 C PRO A 128 8.595 12.637 54.982 1.00 8.34 C ATOM 1852 O PRO A 128 9.390 11.977 54.321 1.00 8.32 O ATOM 1853 N ALA A 129 7.521 13.256 54.480 1.00 7.80 N ATOM 1854 CA ALA A 129 7.226 13.185 53.048 1.00 7.67 C ATOM 1856 CB ALA A 129 6.063 14.115 52.674 1.00 7.71 C ATOM 1860 C ALA A 129 8.476 13.501 52.240 1.00 7.65 C ATOM 1861 O ALA A 129 9.249 14.425 52.549 1.00 7.64 O ATOM 1863 N TRP A 130 8.683 12.696 51.201 1.00 7.53 N ATOM 1864 CA TRP A 130 9.896 12.673 50.390 1.00 7.75 C ATOM 1866 CB TRP A 130 10.025 11.339 49.697 1.00 8.17 C ATOM 1869 CG TRP A 130 10.217 10.140 50.611 1.00 7.44 C ATOM 1870 CD1 TRP A 130 9.246 9.260 51.022 1.00 8.39 C ATOM 1872 NE1 TRP A 130 9.803 8.288 51.843 1.00 8.80 N ATOM 1874 CE2 TRP A 130 11.158 8.509 51.940 1.00 7.48 C ATOM 1875 CD2 TRP A 130 11.456 9.690 51.220 1.00 7.51 C ATOM 1876 CE3 TRP A 130 12.767 10.137 51.175 1.00 9.24 C ATOM 1878 CZ3 TRP A 130 13.729 9.425 51.829 1.00 8.16 C ATOM 1880 CH2 TRP A 130 13.415 8.274 52.564 1.00 9.62 C ATOM 1882 CZ2 TRP A 130 12.139 7.800 52.640 1.00 8.22 C ATOM 1884 C TRP A 130 9.882 13.792 49.350 1.00 8.08 C ATOM 1885 O TRP A 130 9.684 13.553 48.140 1.00 9.57 O ATOM 1887 N MET A 131 10.071 15.011 49.847 1.00 7.97 N ATOM 1888 CA MET A 131 9.981 16.190 49.016 1.00 8.31 C ATOM 1890 CB MET A 131 8.509 16.422 48.584 1.00 9.63 C ATOM 1893 CG MET A 131 7.585 16.674 49.801 1.00 8.68 C ATOM 1896 SD MET A 131 5.828 16.800 49.393 1.00 8.53 S ATOM 1897 CE MET A 131 5.459 15.151 48.784 1.00 8.19 C ATOM 1901 C MET A 131 10.452 17.392 49.823 1.00 8.65 C ATOM 1902 O MET A 131 10.399 17.415 51.058 1.00 8.31 O ATOM 1904 N GLU A 132 10.926 18.409 49.102 1.00 8.97 N ATOM 1905 CA GLU A 132 11.142 19.723 49.712 1.00 8.68 C ATOM 1907 CB GLU A 132 11.613 20.750 48.659 1.00 9.38 C ATOM 1910 CG GLU A 132 12.115 22.055 49.280 1.00 9.41 C ATOM 1913 CD GLU A 132 12.825 22.977 48.300 1.00 12.57 C ATOM 1914 OE1 GLU A 132 12.952 22.614 47.088 1.00 17.82 O ATOM 1915 OE2 GLU A 132 13.203 24.090 48.751 1.00 14.38 O ATOM 1916 C GLU A 132 9.816 20.160 50.334 1.00 9.39 C ATOM 1917 O GLU A 132 8.784 20.126 49.641 1.00 9.43 O ATOM 1919 N ASN A 133 9.876 20.595 51.595 1.00 9.49 N ATOM 1920 CA ASN A 133 8.699 20.897 52.432 1.00 10.02 C ATOM 1922 CB ASN A 133 7.832 21.978 51.781 1.00 11.85 C ATOM 1925 CG ASN A 133 8.611 23.235 51.548 1.00 13.75 C ATOM 1926 OD1 ASN A 133 9.246 23.732 52.480 1.00 19.98 O ATOM 1927 ND2 ASN A 133 8.568 23.774 50.323 1.00 20.37 N ATOM 1930 C ASN A 133 7.952 19.565 52.636 1.00 9.90 C ATOM 1931 O ASN A 133 6.964 19.276 51.936 1.00 10.29 O ATOM 1933 N PRO A 134 8.348 18.784 53.667 1.00 8.19 N ATOM 1934 CA PRO A 134 9.081 19.193 54.857 1.00 8.07 C ATOM 1936 CB PRO A 134 8.806 18.075 55.835 1.00 7.79 C ATOM 1939 CG PRO A 134 8.589 16.870 54.980 1.00 8.16 C ATOM 1942 CD PRO A 134 7.875 17.398 53.765 1.00 7.85 C ATOM 1945 C PRO A 134 10.573 19.393 54.735 1.00 7.98 C ATOM 1946 O PRO A 134 11.143 20.183 55.503 1.00 9.41 O ATOM 1947 N TYR A 135 11.226 18.626 53.867 1.00 7.28 N ATOM 1948 CA TYR A 135 12.685 18.708 53.827 1.00 8.17 C ATOM 1950 CB TYR A 135 13.272 17.691 52.860 1.00 7.94 C ATOM 1953 CG TYR A 135 13.344 16.303 53.449 1.00 7.81 C ATOM 1954 CD1 TYR A 135 14.400 15.969 54.302 1.00 7.46 C ATOM 1956 CE1 TYR A 135 14.494 14.725 54.880 1.00 7.44 C ATOM 1958 CZ TYR A 135 13.546 13.749 54.651 1.00 10.60 C ATOM 1959 OH TYR A 135 13.623 12.511 55.280 1.00 11.23 O ATOM 1961 CE2 TYR A 135 12.467 14.038 53.802 1.00 9.66 C ATOM 1963 CD2 TYR A 135 12.382 15.327 53.205 1.00 8.82 C ATOM 1965 C TYR A 135 13.147 20.120 53.469 1.00 8.11 C ATOM 1966 O TYR A 135 12.553 20.824 52.626 1.00 8.60 O ATOM 1968 N ILE A 136 14.224 20.520 54.144 1.00 8.41 N ATOM 1969 CA ILE A 136 14.860 21.822 53.934 1.00 8.73 C ATOM 1971 CB ILE A 136 15.363 22.435 55.274 1.00 9.60 C ATOM 1973 CG1 ILE A 136 14.201 22.577 56.267 1.00 11.04 C ATOM 1976 CD1 ILE A 136 14.587 22.844 57.736 1.00 13.67 C ATOM 1980 CG2 ILE A 136 16.095 23.749 54.978 1.00 10.67 C ATOM 1984 C ILE A 136 16.032 21.629 52.965 1.00 9.36 C ATOM 1985 O ILE A 136 16.973 20.828 53.217 1.00 8.57 O ATOM 1987 N LYS A 137 15.979 22.326 51.833 1.00 9.57 N ATOM 1988 CA LYS A 137 17.011 22.190 50.828 1.00 10.16 C ATOM 1990 CB LYS A 137 16.547 22.722 49.479 1.00 10.97 C ATOM 1993 CG LYS A 137 17.639 22.695 48.426 1.00 12.27 C ATOM 1996 CD LYS A 137 17.156 23.116 47.044 1.00 14.11 C ATOM 1999 CE LYS A 137 18.242 23.092 46.049 1.00 17.64 C ATOM 2002 NZ LYS A 137 17.759 23.572 44.737 1.00 21.53 N ATOM 2006 C LYS A 137 18.274 22.909 51.282 1.00 10.14 C ATOM 2007 O LYS A 137 18.209 24.117 51.631 1.00 11.61 O ATOM 2009 N VAL A 138 19.391 22.177 51.323 1.00 10.00 N ATOM 2010 CA VAL A 138 20.662 22.750 51.744 1.00 9.75 C ATOM 2012 CB VAL A 138 21.508 21.766 52.568 1.00 10.72 C ATOM 2014 CG1 VAL A 138 22.809 22.438 53.017 1.00 11.63 C ATOM 2018 CG2 VAL A 138 20.746 21.229 53.746 1.00 11.62 C ATOM 2022 C VAL A 138 21.428 23.222 50.523 1.00 10.39 C ATOM 2023 O VAL A 138 21.892 24.373 50.521 1.00 11.53 O ATOM 2025 N ASP A 139 21.580 22.355 49.516 1.00 10.55 N ATOM 2026 CA ASP A 139 22.384 22.691 48.314 1.00 9.26 C ATOM 2028 CB ASP A 139 23.874 22.700 48.644 1.00 9.42 C ATOM 2031 CG ASP A 139 24.738 23.339 47.558 1.00 12.48 C ATOM 2032 OD1 ASP A 139 24.306 24.316 46.912 1.00 15.60 O ATOM 2033 OD2 ASP A 139 25.895 22.865 47.416 1.00 13.71 O ATOM 2034 C ASP A 139 22.166 21.658 47.252 1.00 8.41 C ATOM 2035 O ASP A 139 22.045 20.489 47.573 1.00 9.94 O ATOM 2037 N THR A 140 22.108 22.090 46.003 1.00 9.42 N ATOM 2038 CA THR A 140 22.301 21.170 44.856 1.00 9.48 C ATOM 2040 CB THR A 140 21.510 21.576 43.591 1.00 10.64 C ATOM 2042 OG1 THR A 140 20.096 21.488 43.845 1.00 12.64 O ATOM 2044 CG2 THR A 140 21.857 20.638 42.464 1.00 11.25 C ATOM 2048 C THR A 140 23.809 21.192 44.615 1.00 10.29 C ATOM 2049 O THR A 140 24.403 22.247 44.234 1.00 11.10 O ATOM 2051 N VAL A 141 24.423 20.055 44.914 1.00 9.97 N ATOM 2052 CA VAL A 141 25.890 19.898 44.924 1.00 10.16 C ATOM 2054 CB VAL A 141 26.277 18.801 45.938 1.00 10.62 C ATOM 2056 CG1 VAL A 141 27.799 18.563 45.949 1.00 10.86 C ATOM 2060 CG2 VAL A 141 25.834 19.189 47.341 1.00 10.37 C ATOM 2064 C VAL A 141 26.408 19.547 43.546 1.00 11.24 C ATOM 2065 O VAL A 141 25.976 18.541 42.963 1.00 10.48 O ATOM 2067 N ALA A 142 27.352 20.360 43.056 1.00 11.87 N ATOM 2068 CA ALA A 142 27.983 20.108 41.768 1.00 13.96 C ATOM 2070 CB ALA A 142 28.145 21.383 41.002 1.00 14.94 C ATOM 2074 C ALA A 142 29.318 19.393 41.992 1.00 14.04 C ATOM 2075 O ALA A 142 29.862 19.299 43.121 1.00 14.70 O ATOM 2077 N ALA A 143 29.803 18.761 40.939 1.00 13.39 N ATOM 2078 CA ALA A 143 31.028 18.031 41.014 1.00 12.05 C ATOM 2080 CB ALA A 143 30.843 16.605 40.498 1.00 12.80 C ATOM 2084 C ALA A 143 32.084 18.743 40.182 1.00 12.33 C ATOM 2085 O ALA A 143 31.951 18.796 38.937 1.00 14.44 O ATOM 2087 N GLU A 144 33.139 19.250 40.825 1.00 13.57 N ATOM 2088 CA GLU A 144 34.280 19.809 40.083 1.00 13.75 C ATOM 2090 CB GLU A 144 35.250 20.550 41.014 1.00 16.16 C ATOM 2097 C GLU A 144 35.085 18.722 39.347 1.00 12.44 C ATOM 2098 O GLU A 144 35.769 18.992 38.333 1.00 14.14 O ATOM 2100 N HIS A 145 35.027 17.493 39.860 1.00 10.12 N ATOM 2101 CA HIS A 145 35.796 16.368 39.332 1.00 10.38 C ATOM 2103 CB HIS A 145 36.700 15.784 40.422 1.00 11.19 C ATOM 2106 CG HIS A 145 37.540 16.818 41.088 1.00 14.12 C ATOM 2107 ND1 HIS A 145 38.612 17.412 40.461 1.00 15.31 N ATOM 2109 CE1 HIS A 145 39.149 18.314 41.270 1.00 15.39 C ATOM 2111 NE2 HIS A 145 38.484 18.302 42.410 1.00 15.57 N ATOM 2113 CD2 HIS A 145 37.464 17.374 42.319 1.00 15.78 C ATOM 2115 C HIS A 145 34.901 15.279 38.826 1.00 9.44 C ATOM 2116 O HIS A 145 34.166 14.653 39.601 1.00 9.58 O ATOM 2118 N LEU A 146 34.918 15.065 37.515 1.00 8.62 N ATOM 2119 CA LEU A 146 34.147 13.985 36.905 1.00 8.83 C ATOM 2121 CB LEU A 146 33.532 14.458 35.583 1.00 7.80 C ATOM 2124 CG LEU A 146 32.613 15.647 35.720 1.00 10.46 C ATOM 2126 CD1 LEU A 146 32.213 16.186 34.352 1.00 13.82 C ATOM 2130 CD2 LEU A 146 31.360 15.319 36.532 1.00 12.33 C ATOM 2134 C LEU A 146 35.039 12.782 36.641 1.00 8.88 C ATOM 2135 O LEU A 146 36.196 12.917 36.142 1.00 10.66 O ATOM 2137 N THR A 147 34.519 11.603 36.989 1.00 7.96 N ATOM 2138 CA THR A 147 35.160 10.340 36.676 1.00 8.03 C ATOM 2140 CB THR A 147 34.756 9.277 37.693 1.00 9.05 C ATOM 2142 OG1 THR A 147 35.293 9.664 38.977 1.00 9.24 O ATOM 2144 CG2 THR A 147 35.234 7.926 37.337 1.00 9.73 C ATOM 2148 C THR A 147 34.826 9.923 35.257 1.00 8.52 C ATOM 2149 O THR A 147 33.661 10.037 34.830 1.00 8.12 O ATOM 2151 N ARG A 148 35.858 9.490 34.542 1.00 7.65 N ATOM 2152 CA ARG A 148 35.756 9.129 33.116 1.00 9.81 C ATOM 2154 CB ARG A 148 36.751 9.921 32.277 1.00 10.51 C ATOM 2157 CG ARG A 148 36.501 11.417 32.300 1.00 11.89 C ATOM 2160 CD ARG A 148 37.608 12.217 31.636 1.00 13.56 C ATOM 2163 NE ARG A 148 37.761 11.745 30.264 1.00 18.52 N ATOM 2165 CZ ARG A 148 37.240 12.319 29.175 1.00 17.48 C ATOM 2166 NH1 ARG A 148 36.501 13.464 29.244 1.00 12.11 N ATOM 2169 NH2 ARG A 148 37.438 11.695 27.998 1.00 16.48 N ATOM 2172 C ARG A 148 36.026 7.631 32.940 1.00 10.90 C ATOM 2173 O ARG A 148 36.801 7.072 33.695 1.00 10.86 O ATOM 2175 N LYS A 149 35.376 7.005 31.962 1.00 11.83 N ATOM 2176 CA LYS A 149 35.681 5.589 31.597 1.00 14.09 C ATOM 2178 CB LYS A 149 34.911 4.565 32.415 1.00 15.18 C ATOM 2181 CG LYS A 149 35.164 3.097 31.939 1.00 16.23 C ATOM 2184 CD LYS A 149 34.683 2.035 32.871 1.00 19.77 C ATOM 2187 CE LYS A 149 35.136 0.618 32.360 1.00 17.69 C ATOM 2190 NZ LYS A 149 35.284 0.467 30.843 1.00 22.60 N ATOM 2194 C LYS A 149 35.449 5.411 30.116 1.00 16.14 C ATOM 2195 O LYS A 149 34.402 5.773 29.600 1.00 16.09 O ATOM 2197 N ARG A 150 36.445 4.841 29.449 1.00 16.85 N ATOM 2198 CA ARG A 150 36.590 4.806 27.965 1.00 17.08 C ATOM 2200 CB ARG A 150 37.942 5.349 27.720 1.00 19.68 C ATOM 2203 CG ARG A 150 38.557 4.959 26.571 1.00 19.86 C ATOM 2206 CD ARG A 150 37.760 5.417 25.529 1.00 22.77 C ATOM 2209 NE ARG A 150 37.895 6.784 25.135 1.00 23.77 N ATOM 2211 CZ ARG A 150 38.499 7.765 25.788 1.00 20.28 C ATOM 2212 NH1 ARG A 150 38.461 8.938 25.172 1.00 22.53 N ATOM 2215 NH2 ARG A 150 39.131 7.624 26.975 1.00 18.40 N ATOM 2218 C ARG A 150 36.544 3.358 27.660 1.00 16.33 C ATOM 2219 O ARG A 150 37.001 2.613 28.491 1.00 16.88 O ATOM 2221 N PRO A 151 35.945 2.927 26.503 1.00 15.43 N ATOM 2222 CA PRO A 151 35.669 1.481 26.330 1.00 14.71 C ATOM 2224 CB PRO A 151 35.041 1.386 24.925 1.00 15.62 C ATOM 2227 CG PRO A 151 35.376 2.710 24.211 1.00 15.64 C ATOM 2230 CD PRO A 151 35.407 3.704 25.367 1.00 16.12 C ATOM 2233 C PRO A 151 36.926 0.644 26.427 1.00 14.70 C ATOM 2234 O PRO A 151 37.895 1.013 25.751 1.00 10.80 O ATOM 2235 N GLY A 152 36.885 −0.437 27.224 1.00 11.37 N ATOM 2236 CA GLY A 152 38.053 −1.334 27.452 1.00 13.67 C ATOM 2239 C GLY A 152 38.962 −1.104 28.672 1.00 13.66 C ATOM 2240 O GLY A 152 39.807 −1.950 29.084 1.00 12.78 O ATOM 2242 N ALA A 153 38.745 0.033 29.337 1.00 13.91 N ATOM 2243 CA ALA A 153 39.737 0.621 30.227 1.00 12.51 C ATOM 2245 CB ALA A 153 40.168 1.917 29.612 1.00 11.34 C ATOM 2249 C ALA A 153 39.258 0.882 31.660 1.00 12.62 C ATOM 2250 O ALA A 153 38.043 1.004 31.877 1.00 14.96 O ATOM 2252 N GLU A 154 40.182 0.992 32.614 1.00 12.32 N ATOM 2253 CA GLU A 154 39.825 1.383 33.966 1.00 12.28 C ATOM 2255 CB GLU A 154 40.977 1.233 34.934 1.00 13.61 C ATOM 2262 C GLU A 154 39.329 2.818 34.003 1.00 11.59 C ATOM 2263 O GLU A 154 39.850 3.677 33.311 1.00 9.51 O ATOM 2265 N ALA A 155 38.349 3.064 34.856 1.00 12.64 N ATOM 2266 CA ALA A 155 37.901 4.424 35.135 1.00 11.59 C ATOM 2268 CB ALA A 155 36.676 4.385 36.073 1.00 12.13 C ATOM 2272 C ALA A 155 39.026 5.256 35.750 1.00 11.54 C ATOM 2273 O ALA A 155 39.922 4.759 36.495 1.00 12.39 O ATOM 2275 N THR A 156 39.024 6.567 35.451 1.00 10.29 N ATOM 2276 CA THR A 156 39.991 7.487 36.018 1.00 10.85 C ATOM 2278 CB THR A 156 41.025 8.000 34.960 1.00 12.01 C ATOM 2280 OG1 THR A 156 40.344 8.740 33.942 1.00 16.35 O ATOM 2282 CG2 THR A 156 41.787 6.861 34.355 1.00 14.57 C ATOM 2286 C THR A 156 39.242 8.676 36.572 1.00 11.26 C ATOM 2287 O THR A 156 38.272 9.105 35.972 1.00 11.47 O ATOM 2289 N GLY A 157 39.679 9.210 37.699 1.00 11.15 N ATOM 2290 CA GLY A 157 38.910 10.275 38.306 1.00 11.21 C ATOM 2293 C GLY A 157 39.438 10.644 39.671 1.00 11.02 C ATOM 2294 O GLY A 157 40.298 9.944 40.220 1.00 13.12 O ATOM 2296 N LYS A 158 38.941 11.766 40.139 1.00 10.68 N ATOM 2297 CA LYS A 158 39.181 12.210 41.524 1.00 9.49 C ATOM 2299 CB LYS A 158 39.732 13.623 41.490 1.00 9.39 C ATOM 2302 CG LYS A 158 41.165 13.767 40.912 1.00 10.25 C ATOM 2305 CD LYS A 158 41.557 15.195 40.711 1.00 11.46 C ATOM 2308 CE LYS A 158 43.011 15.314 40.210 1.00 15.64 C ATOM 2311 NZ LYS A 158 43.357 16.712 39.708 1.00 16.95 N ATOM 2315 C LYS A 158 37.913 12.200 42.330 1.00 9.39 C ATOM 2316 O LYS A 158 36.804 12.363 41.805 1.00 9.89 O ATOM 2318 N VAL A 159 38.098 12.081 43.627 1.00 9.10 N ATOM 2319 CA VAL A 159 36.990 12.188 44.564 1.00 9.25 C ATOM 2321 CB VAL A 159 37.323 11.430 45.876 1.00 10.17 C ATOM 2323 CG1 VAL A 159 36.211 11.643 46.920 1.00 11.49 C ATOM 2327 CG2 VAL A 159 37.535 9.939 45.589 1.00 11.58 C ATOM 2331 C VAL A 159 36.674 13.646 44.850 1.00 9.07 C ATOM 2332 O VAL A 159 37.570 14.466 44.949 1.00 10.34 O ATOM 2334 N ASN A 160 35.375 13.967 44.962 1.00 8.44 N ATOM 2335 CA ASN A 160 34.893 15.296 45.314 1.00 7.91 C ATOM 2337 CB ASN A 160 33.565 15.595 44.602 1.00 8.74 C ATOM 2340 CG ASN A 160 33.729 15.628 43.113 1.00 8.92 C ATOM 2341 OD1 ASN A 160 34.114 16.647 42.578 1.00 9.80 O ATOM 2342 ND2 ASN A 160 33.475 14.502 42.443 1.00 10.16 N ATOM 2345 C ASN A 160 34.614 15.359 46.825 1.00 8.46 C ATOM 2346 O ASN A 160 34.234 14.366 47.435 1.00 9.78 O ATOM 2348 N VAL A 161 34.776 16.542 47.378 1.00 8.68 N ATOM 2349 CA VAL A 161 34.398 16.796 48.777 1.00 8.98 C ATOM 2351 CB VAL A 161 35.614 16.836 49.747 1.00 10.70 C ATOM 2353 CG1 VAL A 161 36.624 17.942 49.409 1.00 11.76 C ATOM 2357 CG2 VAL A 161 35.138 16.966 51.192 1.00 9.80 C ATOM 2361 C VAL A 161 33.615 18.117 48.836 1.00 9.38 C ATOM 2362 O VAL A 161 33.955 19.116 48.208 1.00 10.78 O ATOM 2364 N LYS A 162 32.522 18.096 49.594 1.00 9.73 N ATOM 2365 CA LYS A 162 31.708 19.264 49.873 1.00 9.18 C ATOM 2367 CB LYS A 162 30.374 19.207 49.057 1.00 10.14 C ATOM 2370 CG LYS A 162 29.491 20.452 49.235 1.00 13.01 C ATOM 2373 CD LYS A 162 30.092 21.701 48.670 1.00 16.54 C ATOM 2376 CE LYS A 162 29.144 22.929 48.785 1.00 19.66 C ATOM 2379 NZ LYS A 162 29.808 24.238 48.416 1.00 22.42 N ATOM 2383 C LYS A 162 31.361 19.219 51.374 1.00 9.98 C ATOM 2384 O LYS A 162 30.879 18.163 51.860 1.00 10.27 O ATOM 2386 N THR A 163 31.558 20.331 52.077 1.00 9.42 N ATOM 2387 CA THR A 163 31.096 20.469 53.452 1.00 9.80 C ATOM 2389 CB THR A 163 32.246 20.807 54.403 1.00 9.67 C ATOM 2391 OG1 THR A 163 33.214 19.739 54.358 1.00 9.52 O ATOM 2393 CG2 THR A 163 31.765 21.006 55.808 1.00 11.48 C ATOM 2397 C THR A 163 29.992 21.530 53.491 1.00 9.97 C ATOM 2398 O THR A 163 30.157 22.652 52.999 1.00 11.36 O ATOM 2400 N LEU A 164 28.855 21.128 54.042 1.00 10.53 N ATOM 2401 CA LEU A 164 27.671 21.932 54.209 1.00 10.36 C ATOM 2403 CB LEU A 164 26.474 21.165 53.625 1.00 11.41 C ATOM 2406 CG LEU A 164 26.646 20.822 52.143 1.00 13.33 C ATOM 2408 CD1 LEU A 164 25.497 19.943 51.643 1.00 16.96 C ATOM 2412 CD2 LEU A 164 26.756 22.072 51.298 1.00 16.24 C ATOM 2416 C LEU A 164 27.440 22.268 55.670 1.00 10.61 C ATOM 2417 O LEU A 164 27.750 21.473 56.555 1.00 12.15 O ATOM 2419 N ARG A 165 26.914 23.463 55.924 1.00 10.93 N ATOM 2420 CA ARG A 165 26.586 23.874 57.284 1.00 11.96 C ATOM 2422 CB ARG A 165 27.171 25.240 57.599 1.00 12.49 C ATOM 2425 CG ARG A 165 26.974 25.703 59.029 1.00 13.26 C ATOM 2428 CD ARG A 165 27.616 27.089 59.136 1.00 15.90 C ATOM 2431 NE ARG A 165 27.727 27.688 60.469 1.00 20.51 N ATOM 2433 CZ ARG A 165 27.136 28.826 60.844 1.00 21.93 C ATOM 2434 NH1 ARG A 165 26.352 29.490 60.013 1.00 23.30 N ATOM 2437 NH2 ARG A 165 27.315 29.303 62.067 1.00 23.95 N ATOM 2440 C ARG A 165 25.056 23.909 57.443 1.00 12.67 C ATOM 2441 O ARG A 165 24.345 24.577 56.664 1.00 14.74 O ATOM 2443 N LEU A 166 24.577 23.178 58.436 1.00 11.74 N ATOM 2444 CA LEU A 166 23.132 22.964 58.690 1.00 11.51 C ATOM 2446 CB LEU A 166 22.792 21.486 58.930 1.00 12.12 C ATOM 2449 CG LEU A 166 23.316 20.409 58.006 1.00 17.08 C ATOM 2451 CD1 LEU A 166 22.908 19.019 58.467 1.00 19.61 C ATOM 2455 CD2 LEU A 166 22.886 20.648 56.641 1.00 20.41 C ATOM 2459 C LEU A 166 22.748 23.732 59.945 1.00 11.98 C ATOM 2460 O LEU A 166 23.450 23.645 60.930 1.00 12.34 O ATOM 2462 N GLY A 167 21.563 24.386 59.937 1.00 12.12 N ATOM 2463 CA GLY A 167 21.048 25.052 61.106 1.00 12.77 C ATOM 2466 C GLY A 167 20.628 26.496 60.813 1.00 14.49 C ATOM 2467 O GLY A 167 20.639 26.913 59.642 1.00 14.88 O ATOM 2469 N PRO A 168 20.256 27.233 61.861 1.00 14.26 N ATOM 2470 CA PRO A 168 20.320 26.798 63.252 1.00 13.89 C ATOM 2472 CB PRO A 168 20.073 28.093 64.030 1.00 15.57 C ATOM 2475 CG PRO A 168 19.159 28.871 63.098 1.00 16.62 C ATOM 2478 CD PRO A 168 19.658 28.586 61.740 1.00 15.12 C ATOM 2481 C PRO A 168 19.276 25.749 63.616 1.00 13.01 C ATOM 2482 O PRO A 168 18.112 25.762 63.108 1.00 14.29 O ATOM 2483 N LEU A 169 19.698 24.816 64.458 1.00 12.79 N ATOM 2484 CA LEU A 169 18.906 23.655 64.842 1.00 11.32 C ATOM 2486 CB LEU A 169 19.767 22.400 64.948 1.00 12.93 C ATOM 2489 CG LEU A 169 20.561 22.078 63.691 1.00 13.60 C ATOM 2491 CD1 LEU A 169 21.509 20.931 64.018 1.00 16.57 C ATOM 2495 CD2 LEU A 169 19.626 21.728 62.550 1.00 15.53 C ATOM 2499 C LEU A 169 18.244 23.882 66.193 1.00 13.18 C ATOM 2500 O LEU A 169 18.778 24.595 67.052 1.00 13.33 O ATOM 2502 N SER A 170 17.052 23.329 66.370 1.00 12.97 N ATOM 2503 CA SER A 170 16.352 23.474 67.646 1.00 14.51 C ATOM 2505 CB SER A 170 15.393 24.637 67.507 1.00 15.01 C ATOM 2508 OG SER A 170 14.303 24.316 66.678 1.00 22.23 O ATOM 2510 C SER A 170 15.600 22.251 68.173 1.00 13.02 C ATOM 2511 O SER A 170 15.258 22.199 69.366 1.00 17.05 O ATOM 2513 N LYS A 171 15.368 21.242 67.327 1.00 11.31 N ATOM 2514 CA LYS A 171 14.722 20.011 67.784 1.00 10.60 C ATOM 2516 CB LYS A 171 13.782 19.493 66.691 1.00 10.19 C ATOM 2519 CG LYS A 171 12.594 20.391 66.388 1.00 11.75 C ATOM 2522 CD LYS A 171 11.530 19.673 65.517 1.00 11.58 C ATOM 2525 CE LYS A 171 10.212 20.409 65.393 1.00 14.91 C ATOM 2528 NZ LYS A 171 9.250 19.814 64.374 1.00 15.60 N ATOM 2532 C LYS A 171 15.732 18.959 68.255 1.00 10.82 C ATOM 2533 O LYS A 171 16.956 19.151 68.092 1.00 12.33 O ATOM 2535 N ALA A 172 15.274 17.855 68.823 1.00 9.49 N ATOM 2536 CA ALA A 172 16.192 16.873 69.457 1.00 8.93 C ATOM 2538 CB ALA A 172 15.405 15.776 70.239 1.00 9.36 C ATOM 2542 C ALA A 172 17.180 16.212 68.488 1.00 9.88 C ATOM 2543 O ALA A 172 18.304 15.868 68.911 1.00 11.09 O ATOM 2545 N GLY A 173 16.756 16.017 67.245 1.00 9.34 N ATOM 2546 CA GLY A 173 17.600 15.356 66.238 1.00 9.33 C ATOM 2549 C GLY A 173 17.131 15.694 64.837 1.00 9.73 C ATOM 2550 O GLY A 173 16.217 16.515 64.646 1.00 9.04 O ATOM 2552 N PHE A 174 17.827 15.117 63.865 1.00 7.56 N ATOM 2553 CA PHE A 174 17.552 15.372 62.466 1.00 7.53 C ATOM 2555 CB PHE A 174 18.263 16.652 61.997 1.00 8.21 C ATOM 2558 CG PHE A 174 19.749 16.581 62.118 1.00 8.24 C ATOM 2559 CD1 PHE A 174 20.523 16.170 61.026 1.00 10.68 C ATOM 2561 CE1 PHE A 174 21.904 16.075 61.163 1.00 11.00 C ATOM 2563 CZ PHE A 174 22.494 16.378 62.367 1.00 9.97 C ATOM 2565 CE2 PHE A 174 21.770 16.762 63.426 1.00 11.65 C ATOM 2567 CD2 PHE A 174 20.374 16.875 63.317 1.00 11.58 C ATOM 2569 C PHE A 174 17.936 14.203 61.550 1.00 8.39 C ATOM 2570 O PHE A 174 18.735 13.329 61.906 1.00 9.32 O ATOM 2572 N TYR A 175 17.336 14.228 60.359 1.00 8.25 N ATOM 2573 CA TYR A 175 17.658 13.316 59.259 1.00 7.48 C ATOM 2575 CB TYR A 175 16.413 12.637 58.676 1.00 9.30 C ATOM 2578 CG TYR A 175 15.584 11.889 59.688 1.00 7.58 C ATOM 2579 CD1 TYR A 175 15.835 10.546 59.969 1.00 8.86 C ATOM 2581 CE1 TYR A 175 15.095 9.842 60.902 1.00 9.03 C ATOM 2583 CZ TYR A 175 14.030 10.433 61.508 1.00 8.70 C ATOM 2584 OH TYR A 175 13.278 9.774 62.464 1.00 8.58 O ATOM 2586 CE2 TYR A 175 13.757 11.778 61.240 1.00 9.04 C ATOM 2588 CD2 TYR A 175 14.519 12.480 60.322 1.00 9.76 C ATOM 2590 C TYR A 175 18.321 14.102 58.154 1.00 9.02 C ATOM 2591 O TYR A 175 17.914 15.248 57.902 1.00 9.13 O ATOM 2593 N LEU A 176 19.326 13.515 57.485 1.00 9.33 N ATOM 2594 CA LEU A 176 19.782 14.072 56.220 1.00 10.45 C ATOM 2596 CB LEU A 176 21.319 14.087 56.124 1.00 11.50 C ATOM 2599 CG LEU A 176 22.065 15.260 55.571 1.00 19.01 C ATOM 2601 CD1 LEU A 176 21.729 16.554 56.106 1.00 24.20 C ATOM 2605 CD2 LEU A 176 23.588 15.037 55.662 1.00 16.96 C ATOM 2609 C LEU A 176 19.192 13.224 55.106 1.00 10.60 C ATOM 2610 O LEU A 176 19.009 11.996 55.285 1.00 11.03 O ATOM 2612 N ALA A 177 18.874 13.828 53.956 1.00 9.29 N ATOM 2613 CA ALA A 177 18.364 13.067 52.816 1.00 9.08 C ATOM 2615 CB ALA A 177 16.871 13.142 52.676 1.00 10.99 C ATOM 2619 C ALA A 177 19.051 13.561 51.558 1.00 10.04 C ATOM 2620 O ALA A 177 19.398 14.737 51.438 1.00 9.75 O ATOM 2622 N PHE A 178 19.222 12.627 50.630 1.00 9.02 N ATOM 2623 CA PHE A 178 19.846 12.880 49.346 1.00 9.63 C ATOM 2625 CB PHE A 178 21.096 12.010 49.256 1.00 9.71 C ATOM 2628 CG PHE A 178 22.014 12.180 50.397 1.00 11.34 C ATOM 2629 CD1 PHE A 178 22.904 13.231 50.406 1.00 17.04 C ATOM 2631 CE1 PHE A 178 23.766 13.393 51.473 1.00 19.52 C ATOM 2633 CZ PHE A 178 23.687 12.533 52.567 1.00 15.86 C ATOM 2635 CE2 PHE A 178 22.828 11.493 52.563 1.00 12.43 C ATOM 2637 CD2 PHE A 178 21.968 11.319 51.505 1.00 11.87 C ATOM 2639 C PHE A 178 18.884 12.509 48.243 1.00 10.24 C ATOM 2640 O PHE A 178 18.310 11.419 48.264 1.00 10.41 O ATOM 2642 N GLN A 179 18.706 13.407 47.281 1.00 9.37 N ATOM 2643 CA GLN A 179 17.882 13.172 46.105 1.00 9.39 C ATOM 2645 CB GLN A 179 16.841 14.275 45.937 1.00 9.56 C ATOM 2648 CG GLN A 179 15.926 14.032 44.737 1.00 10.70 C ATOM 2651 CD GLN A 179 15.123 15.238 44.384 1.00 14.67 C ATOM 2652 OE1 GLN A 179 15.614 16.371 44.426 1.00 19.87 O ATOM 2653 NE2 GLN A 179 13.835 15.015 44.063 1.00 19.39 N ATOM 2656 C GLN A 179 18.780 13.131 44.862 1.00 9.70 C ATOM 2657 O GLN A 179 19.507 14.063 44.582 1.00 9.12 O ATOM 2659 N ASP A 180 18.681 12.017 44.151 1.00 9.12 N ATOM 2660 CA ASP A 180 19.246 11.827 42.834 1.00 8.67 C ATOM 2662 CB ASP A 180 19.988 10.495 42.808 1.00 9.62 C ATOM 2665 CG ASP A 180 20.230 10.004 41.384 1.00 11.84 C ATOM 2666 OD1 ASP A 180 21.098 10.598 40.700 1.00 9.57 O ATOM 2667 OD2 ASP A 180 19.506 9.069 40.929 1.00 10.67 O ATOM 2668 C ASP A 180 18.165 11.825 41.811 1.00 9.18 C ATOM 2669 O ASP A 180 17.071 11.293 42.046 1.00 9.26 O ATOM 2671 N GLN A 181 18.429 12.401 40.653 1.00 7.59 N ATOM 2672 CA GLN A 181 17.486 12.287 39.502 1.00 9.62 C ATOM 2674 CB GLN A 181 16.446 13.404 39.553 1.00 9.54 C ATOM 2677 CG GLN A 181 15.294 13.192 38.629 1.00 12.83 C ATOM 2680 CD GLN A 181 14.240 14.249 38.781 1.00 17.44 C ATOM 2681 OE1 GLN A 181 13.142 13.998 39.296 1.00 25.22 O ATOM 2682 NE2 GLN A 181 14.536 15.437 38.295 1.00 24.11 N ATOM 2685 C GLN A 181 18.323 12.339 38.220 1.00 9.56 C ATOM 2686 O GLN A 181 18.688 13.405 37.760 1.00 10.29 O ATOM 2688 N GLY A 182 18.706 11.170 37.715 1.00 9.26 N ATOM 2689 CA GLY A 182 19.458 11.066 36.463 1.00 9.98 C ATOM 2692 C GLY A 182 20.962 11.281 36.539 1.00 9.35 C ATOM 2693 O GLY A 182 21.615 11.486 35.523 1.00 9.38 O ATOM 2695 N ALA A 183 21.531 11.165 37.740 1.00 8.87 N ATOM 2696 CA ALA A 183 22.990 11.245 37.923 1.00 8.24 C ATOM 2698 CB ALA A 183 23.362 12.143 39.062 1.00 8.69 C ATOM 2702 C ALA A 183 23.548 9.855 38.159 1.00 8.99 C ATOM 2703 O ALA A 183 22.798 8.874 38.249 1.00 10.60 O ATOM 2705 N CYS A 184 24.874 9.772 38.195 1.00 7.92 N ATOM 2706 CA CYS A 184 25.549 8.477 38.316 1.00 7.85 C ATOM 2708 CB CYS A 184 26.054 8.023 36.956 1.00 8.58 C ATOM 2711 SG CYS A 184 26.830 6.361 36.962 1.00 9.83 S ATOM 2713 C CYS A 184 26.704 8.693 39.292 1.00 7.08 C ATOM 2714 O CYS A 184 27.735 9.254 38.916 1.00 8.95 O ATOM 2716 N MET A 185 26.516 8.289 40.536 1.00 7.67 N ATOM 2717 CA MET A 185 27.400 8.698 41.620 1.00 7.79 C ATOM 2719 CB MET A 185 26.891 9.986 42.242 1.00 8.31 C ATOM 2722 CG MET A 185 25.509 9.869 42.942 1.00 8.09 C ATOM 2725 SD MET A 185 25.067 11.370 43.893 1.00 10.10 S ATOM 2726 CE MET A 185 26.262 11.292 45.178 1.00 11.25 C ATOM 2730 C MET A 185 27.555 7.657 42.707 1.00 8.04 C ATOM 2731 O MET A 185 26.716 6.768 42.891 1.00 7.27 O ATOM 2733 N ALA A 186 28.681 7.761 43.406 1.00 7.98 N ATOM 2734 CA ALA A 186 28.926 6.985 44.617 1.00 9.39 C ATOM 2736 CB ALA A 186 30.175 6.146 44.499 1.00 8.65 C ATOM 2740 C ALA A 186 29.118 7.945 45.786 1.00 9.09 C ATOM 2741 O ALA A 186 30.032 8.789 45.742 1.00 8.21 O ATOM 2743 N LEU A 187 28.282 7.814 46.817 1.00 8.78 N ATOM 2744 CA LEU A 187 28.487 8.519 48.112 1.00 9.03 C ATOM 2746 CB LEU A 187 27.152 8.751 48.826 1.00 9.73 C ATOM 2749 CG LEU A 187 27.259 9.515 50.135 1.00 11.78 C ATOM 2751 CD1 LEU A 187 27.747 10.946 49.895 1.00 13.80 C ATOM 2755 CD2 LEU A 187 25.896 9.537 50.804 1.00 15.87 C ATOM 2759 C LEU A 187 29.421 7.629 48.946 1.00 8.46 C ATOM 2760 O LEU A 187 29.051 6.559 49.435 1.00 8.87 O ATOM 2762 N LEU A 188 30.666 8.065 49.036 1.00 8.40 N ATOM 2763 CA LEU A 188 31.722 7.307 49.652 1.00 8.29 C ATOM 2765 CB LEU A 188 33.071 7.652 49.003 1.00 9.53 C ATOM 2768 CG LEU A 188 33.166 7.411 47.482 1.00 9.10 C ATOM 2770 CD1 LEU A 188 34.477 7.931 46.911 1.00 11.01 C ATOM 2774 CD2 LEU A 188 33.024 5.950 47.150 1.00 11.04 C ATOM 2778 C LEU A 188 31.797 7.477 51.146 1.00 8.56 C ATOM 2779 O LEU A 188 32.177 6.551 51.836 1.00 9.83 O ATOM 2781 N SER A 189 31.428 8.647 51.620 1.00 8.94 N ATOM 2782 CA SER A 189 31.360 8.899 53.068 1.00 8.72 C ATOM 2784 CB SER A 189 32.757 8.981 53.658 1.00 10.49 C ATOM 2787 OG SER A 189 33.482 10.025 53.106 1.00 12.31 O ATOM 2789 C SER A 189 30.585 10.154 53.420 1.00 8.61 C ATOM 2790 O SER A 189 30.426 11.093 52.632 1.00 8.28 O ATOM 2792 N LEU A 190 29.981 10.099 54.613 1.00 8.03 N ATOM 2793 CA LEU A 190 29.319 11.229 55.254 1.00 7.40 C ATOM 2795 CB LEU A 190 27.778 11.045 55.319 1.00 7.78 C ATOM 2798 CG LEU A 190 27.038 12.050 56.182 1.00 8.27 C ATOM 2800 CD1 LEU A 190 27.148 13.503 55.675 1.00 10.17 C ATOM 2804 CD2 LEU A 190 25.540 11.644 56.344 1.00 8.94 C ATOM 2808 C LEU A 190 29.895 11.315 56.674 1.00 8.35 C ATOM 2809 O LEU A 190 29.892 10.323 57.437 1.00 8.74 O ATOM 2811 N HIS A 191 30.306 12.512 57.072 1.00 7.60 N ATOM 2812 CA HIS A 191 30.745 12.781 58.457 1.00 7.90 C ATOM 2814 CB HIS A 191 32.260 12.962 58.452 1.00 8.03 C ATOM 2817 CG HIS A 191 32.910 12.968 59.808 1.00 8.71 C ATOM 2818 ND1 HIS A 191 34.281 12.895 59.947 1.00 11.85 N ATOM 2820 CE1 HIS A 191 34.588 12.927 61.239 1.00 11.49 C ATOM 2822 NE2 HIS A 191 33.467 13.013 61.940 1.00 8.44 N ATOM 2824 CD2 HIS A 191 32.403 13.030 61.069 1.00 8.15 C ATOM 2826 C HIS A 191 30.071 14.048 58.935 1.00 8.00 C ATOM 2827 O HIS A 191 30.204 15.094 58.323 1.00 9.66 O ATOM 2829 N LEU A 192 29.224 13.867 59.943 1.00 8.43 N ATOM 2830 CA LEU A 192 28.549 14.963 60.619 1.00 9.08 C ATOM 2832 CB LEU A 192 27.099 14.577 60.975 1.00 10.10 C ATOM 2835 CG LEU A 192 26.167 14.276 59.809 1.00 11.69 C ATOM 2837 CD1 LEU A 192 24.869 13.680 60.288 1.00 12.31 C ATOM 2841 CD2 LEU A 192 25.899 15.492 59.016 1.00 13.67 C ATOM 2845 C LEU A 192 29.293 15.297 61.893 1.00 7.94 C ATOM 2846 O LEU A 192 29.645 14.397 62.676 1.00 8.07 O ATOM 2848 N PHE A 193 29.556 16.583 62.131 1.00 8.07 N ATOM 2849 CA PHE A 193 30.262 17.006 63.330 1.00 7.59 C ATOM 2851 CB PHE A 193 31.797 16.907 63.174 1.00 7.55 C ATOM 2854 CG PHE A 193 32.371 17.852 62.160 1.00 9.51 C ATOM 2855 CD1 PHE A 193 32.741 19.151 62.540 1.00 9.02 C ATOM 2857 CE1 PHE A 193 32.253 20.032 61.585 1.00 9.94 C ATOM 2859 CZ PHE A 193 33.398 19.610 60.285 1.00 8.63 C ATOM 2861 CE2 PHE A 193 33.023 18.360 59.896 1.00 11.72 C ATOM 2863 CD2 PHE A 193 32.508 17.471 60.815 1.00 10.23 C ATOM 2865 C PHE A 193 29.835 18.415 63.705 1.00 8.62 C ATOM 2866 O PHE A 193 29.228 19.118 62.894 1.00 8.78 O ATOM 2868 N TYR A 194 30.141 18.800 64.938 1.00 8.07 N ATOM 2869 CA TYR A 194 29.910 20.193 65.386 1.00 8.73 C ATOM 2871 CB TYR A 194 28.589 20.329 66.175 1.00 9.62 C ATOM 2874 CG TYR A 194 28.510 19.615 67.517 1.00 8.79 C ATOM 2875 CD1 TYR A 194 28.578 20.311 68.731 1.00 11.90 C ATOM 2877 CE1 TYR A 194 28.489 19.623 69.948 1.00 12.30 C ATOM 2879 CZ TYR A 194 28.319 18.252 69.969 1.00 13.69 C ATOM 2880 OH TYR A 194 28.213 17.570 71.188 1.00 16.83 O ATOM 2882 CE2 TYR A 194 28.269 17.553 68.770 1.00 13.74 C ATOM 2884 CD2 TYR A 194 28.363 18.232 67.581 1.00 10.43 C ATOM 2886 C TYR A 194 31.068 20.698 66.219 1.00 9.43 C ATOM 2887 O TYR A 194 31.923 19.929 66.662 1.00 9.21 O ATOM 2889 N LYS A 195 31.104 22.018 66.437 1.00 10.08 N ATOM 2890 CA LYS A 195 32.157 22.657 67.262 1.00 11.96 C ATOM 2892 CB LYS A 195 32.548 24.044 66.674 1.00 13.33 C ATOM 2899 C LYS A 195 31.648 22.754 68.692 1.00 12.87 C ATOM 2900 O LYS A 195 30.573 23.352 68.946 1.00 12.94 O ATOM 2902 N LYS A 196 32.383 22.113 69.611 1.00 14.64 N ATOM 2903 CA LYS A 196 32.037 22.049 71.058 1.00 16.38 C ATOM 2905 CB LYS A 196 32.025 20.593 71.561 1.00 16.65 C ATOM 2908 CG LYS A 196 31.247 20.318 72.849 1.00 19.41 C ATOM 2911 CD LYS A 196 31.368 18.853 73.224 1.00 20.76 C ATOM 2914 CE LYS A 196 30.555 18.496 74.506 1.00 21.67 C ATOM 2917 NZ LYS A 196 31.158 18.990 75.740 1.00 26.89 N ATOM 2921 C LYS A 196 33.054 22.847 71.831 1.00 17.46 C ATOM 2922 O LYS A 196 34.262 22.740 71.576 1.00 17.70 O ATOM 2924 N ASN A 251 43.160 4.312 44.067 1.00 22.03 N ATOM 2925 CA ASN A 251 42.141 5.288 44.481 1.00 21.06 C ATOM 2927 CB ASN A 251 42.373 6.675 43.819 1.00 21.54 C ATOM 2930 CG ASN P 251 41.459 7.820 44.415 1.00 23.63 C ATOM 2931 OD1 ASN P 251 41.322 8.907 43.808 1.00 25.42 O ATOM 2932 ND2 ASN P 251 40.886 7.585 45.615 1.00 22.93 N ATOM 2935 C ASN P 251 40.751 4.645 44.149 1.00 19.88 C ATOM 2936 O ASN P 251 40.317 4.563 42.982 1.00 21.13 O ATOM 2940 N TYR P 252 40.098 4.148 45.183 1.00 15.93 N ATOM 2941 CA TYR P 252 38.770 3.589 45.052 1.00 14.57 C ATOM 2943 CB TYR P 252 38.328 2.996 46.409 1.00 14.90 C ATOM 2946 CG TYR P 252 36.982 2.348 46.341 1.00 13.17 C ATOM 2947 CD1 TYR P 252 36.840 0.998 45.980 1.00 15.56 C ATOM 2949 CE1 TYR P 252 35.610 0.399 45.879 1.00 14.96 C ATOM 2951 CZ TYR P 252 34.472 1.148 46.129 1.00 15.22 C ATOM 2952 OH TYR P 252 33.208 0.610 46.073 1.00 13.93 O ATOM 2954 CE2 TYR P 252 34.589 2.455 46.537 1.00 14.09 C ATOM 2956 CD2 TYR P 252 35.852 3.053 46.616 1.00 13.95 C ATOM 2958 C TYR P 252 37.771 4.690 44.624 1.00 13.53 C ATOM 2959 O TYR P 252 37.636 5.714 45.317 1.00 14.00 O ATOM 2961 N LEU P 253 37.072 4.461 43.522 1.00 12.91 N ATOM 2962 CA LEU P 253 36.060 5.416 43.022 1.00 11.40 C ATOM 2964 CB LEU P 253 36.359 5.843 41.580 1.00 11.85 C ATOM 2967 CG LEU P 253 37.702 6.574 41.405 1.00 11.52 C ATOM 2969 CD1 LEU P 253 38.051 6.861 39.938 1.00 14.14 C ATOM 2973 CD2 LEU P 253 37.735 7.838 42.176 1.00 13.77 C ATOM 2977 C LEU P 253 34.642 4.844 43.115 1.00 12.69 C ATOM 2978 O LEU P 253 33.716 5.576 43.386 1.00 13.84 O ATOM 2980 N PHE P 254 34.471 3.549 42.831 1.00 11.86 N ATOM 2981 CA PHE P 254 33.198 2.865 42.874 1.00 12.02 C ATOM 2983 CB PHE P 254 32.261 3.307 41.741 1.00 13.21 C ATOM 2986 CG PHE P 254 32.806 3.116 40.346 1.00 13.27 C ATOM 2987 CD1 PHE P 254 32.742 1.887 39.712 1.00 15.51 C ATOM 2989 CE1 PHE P 254 33.218 1.718 38.441 1.00 17.28 C ATOM 2991 CZ PHE P 254 33.784 2.777 37.760 1.00 18.13 C ATOM 2993 CE2 PHE P 254 33.848 4.025 38.396 1.00 17.44 C ATOM 2995 CD2 PHE P 254 33.380 4.176 39.674 1.00 15.88 C ATOM 2997 C PHE P 254 33.416 1.353 42.848 1.00 11.55 C ATOM 2998 O PHE P 254 34.547 0.864 42.674 1.00 12.47 O ATOM 3000 N SER P 255 32.317 0.633 43.050 1.00 13.41 N ATOM 3001 CA SER P 255 32.350 −0.813 43.277 1.00 13.05 C ATOM 3003 CB SER P 255 31.111 −1.266 44.041 1.00 13.36 C ATOM 3006 OG SER P 255 30.995 −2.681 44.116 1.00 14.10 O ATOM 3008 C SER P 255 32.362 −1.565 41.971 1.00 14.28 C ATOM 3009 O SER P 255 31.717 −1.153 41.010 1.00 15.30 O ATOM 3011 N PRO P 256 33.069 −2.701 41.933 1.00 14.39 N ATOM 3012 CA PRO P 256 32.933 −3.542 40.748 1.00 14.47 C ATOM 3014 CB PRO P 256 34.011 −4.593 40.928 1.00 15.06 C ATOM 3017 CG PRO P 256 34.313 −4.609 42.373 1.00 15.97 C ATOM 3020 CD PRO P 256 33.979 −3.265 42.941 1.00 15.21 C ATOM 3023 C PRO P 256 31.574 −4.220 40.607 1.00 14.15 C ATOM 3024 O PRO P 256 31.268 −4.758 39.529 1.00 14.96 O ATOM 3025 N ASN P 257 30.767 −4.179 41.655 1.00 13.76 N ATOM 3026 CA ASN P 257 29.495 −4.927 41.742 1.00 14.91 C ATOM 3028 CB ASN P 257 29.528 −5.761 43.019 1.00 16.24 C ATOM 3031 CG ASN P 257 30.710 −6.703 43.075 1.00 19.43 C ATOM 3032 OD1 ASN P 257 31.147 −7.233 42.056 1.00 19.88 O ATOM 3033 ND2 ASN P 257 31.217 −6.940 44.283 1.00 22.73 N ATOM 3036 C ASN P 257 28.233 −4.088 41.832 1.00 14.65 C ATOM 3037 O ASN P 257 27.166 −4.633 42.116 1.00 14.56 O ATOM 3039 N GLY P 258 28.349 −2.782 41.612 1.00 13.71 N ATOM 3040 CA GLY P 258 27.226 −1.869 41.782 1.00 13.39 C ATOM 3043 C GLY P 258 26.633 −1.319 40.490 1.00 13.17 C ATOM 3044 O GLY P 258 26.963 −1.794 39.408 1.00 13.88 O ATOM 3046 N PRO P 259 25.669 −0.356 40.634 1.00 13.28 N ATOM 3047 CA PRO P 259 24.924 0.159 39.482 1.00 13.62 C ATOM 3049 CB PRO P 259 23.826 1.046 40.106 1.00 14.38 C ATOM 3052 CG PRO P 259 24.209 1.284 41.465 1.00 14.55 C ATOM 3055 CD PRO P 259 25.169 0.197 41.902 1.00 14.06 C ATOM 3058 C PRO P 259 25.791 0.979 38.544 1.00 13.22 C ATOM 3059 O PRO P 259 25.481 1.101 37.327 1.00 13.10 O ATOM 3060 N ILE P 260 26.879 1.524 39.081 1.00 13.54 N ATOM 3061 CA ILE P 260 27.816 2.249 38.235 1.00 13.31 C ATOM 3063 CB ILE P 260 28.826 3.067 39.067 1.00 12.69 C ATOM 3065 CG1 ILE P 260 28.069 4.128 39.881 1.00 12.98 C ATOM 3068 CD1 ILE P 260 28.883 4.824 40.935 1.00 14.32 C ATOM 3072 CG2 ILE P 260 29.810 3.759 38.138 1.00 13.16 C ATOM 3076 C ILE P 260 28.538 1.293 37.279 1.00 13.17 C ATOM 3077 O ILE P 260 28.536 1.480 36.056 1.00 12.37 O ATOM 3079 N ALA P 261 29.132 0.236 37.826 1.00 13.12 N ATOM 3080 CA ALA P 261 29.780 −0.765 36.998 1.00 13.37 C ATOM 3082 CB ALA P 261 30.371 −1.875 37.861 1.00 14.02 C ATOM 3086 C ALA P 261 28.830 −1.344 35.955 1.00 14.14 C ATOM 3087 O ALA P 261 29.239 −1.651 34.831 1.00 15.28 O ATOM 3089 N ARG P 262 27.566 −1.517 36.326 1.00 13.51 N ATOM 3090 CA ARG P 262 26.558 −2.069 35.421 1.00 14.66 C ATOM 3092 CB ARG P 262 25.272 −2.428 36.205 1.00 15.22 C ATOM 3095 CG ARG P 262 25.455 −3.668 37.078 1.00 16.13 C ATOM 3098 CD ARG P 262 24.124 −4.259 37.537 1.00 16.60 C ATOM 3101 NE ARG P 262 23.470 −3.395 38.513 1.00 17.89 N ATOM 3103 CZ ARG P 262 23.689 −3.419 39.816 1.00 18.17 C ATOM 3104 NH1 ARG P 262 24.573 −4.225 40.375 1.00 17.86 N ATOM 3107 NH2 ARG P 262 23.025 −2.582 40.577 1.00 20.47 N ATOM 3110 C ARG P 262 26.221 −1.156 34.242 1.00 15.24 C ATOM 3111 O ARG P 262 25.629 −1.634 33.239 1.00 15.81 O ATOM 3113 N ALA P 263 26.605 0.121 34.320 1.00 16.39 N ATOM 3114 CA ALA P 263 26.379 1.030 33.211 1.00 17.38 C ATOM 3116 CB ALA P 263 26.783 2.445 33.547 1.00 17.32 C ATOM 3120 C ALA P 263 27.118 0.550 31.947 1.00 18.17 C ATOM 3121 O ALA P 263 26.744 0.929 30.831 1.00 18.92 O ATOM 3123 N TRP P 264 28.202 −0.202 32.111 1.00 18.44 N ATOM 3124 CA TRP P 264 29.019 −0.654 30.952 1.00 19.37 C ATOM 3126 CB TRP P 264 30.510 −0.370 31.200 1.00 20.30 C ATOM 3129 CG TRP P 264 30.730 1.090 31.441 1.00 20.52 C ATOM 3130 CD1 TRP P 264 30.626 2.093 30.530 1.00 24.02 C ATOM 3132 NE1 TRP P 264 30.859 3.305 31.129 1.00 21.70 N ATOM 3134 CE2 TRP P 264 31.143 3.103 32.440 1.00 23.96 C ATOM 3135 CD2 TRP P 264 31.054 1.711 32.678 1.00 20.21 C ATOM 3136 CE3 TRP P 264 31.337 1.231 33.950 1.00 21.14 C ATOM 3138 CZ3 TRP P 264 31.642 2.143 34.959 1.00 23.62 C ATOM 3140 CH2 TRP P 264 31.684 3.522 34.702 1.00 23.52 C ATOM 3142 CZ2 TRP P 264 31.423 4.019 33.450 1.00 20.98 C ATOM 3144 C TRP P 264 28.773 −2.117 30.645 1.00 20.85 C ATOM 3145 O TRP P 264 27.899 −2.714 31.272 1.00 22.43 O ATOM 3147 OXT TRP P 264 29.405 −2.712 29.746 1.00 21.74 O ATOM 3148 O HOH X 301 34.430 11.979 40.417 1.00 7.01 O ATOM 3151 O HOH X 302 8.396 11.854 46.295 1.00 10.84 O ATOM 3154 O HOH X 304 31.629 5.424 54.367 1.00 7.78 O ATOM 3157 O HOH X 305 29.393 0.001 40.716 1.00 11.67 O ATOM 3160 O HOH X 306 26.504 11.508 36.590 1.00 11.86 O ATOM 3163 O HOH X 308 31.743 1.752 56.936 1.00 11.18 O ATOM 3166 O HOH X 309 5.324 13.803 56.433 1.00 10.31 O ATOM 3169 O HOH X 310 10.666 3.747 51.782 1.00 11.18 O ATOM 3172 O HOH X 311 8.900 13.123 61.479 1.00 17.18 O ATOM 3175 O HOH X 312 11.707 10.700 54.483 1.00 6.76 O ATOM 3178 O HOH X 313 15.905 6.703 58.672 1.00 13.28 O ATOM 3181 O HOH X 314 35.962 18.454 45.448 1.00 12.13 O ATOM 3184 O HOH X 315 4.724 20.141 50.518 1.00 13.74 O ATOM 3187 O HOH X 316 20.949 2.851 39.943 1.00 12.79 O ATOM 3190 O HOH X 317 17.280 8.646 37.970 1.00 11.75 O ATOM 3193 O HOH X 318 32.871 12.284 54.535 1.00 8.88 O ATOM 3196 O HOH X 319 38.982 4.909 31.022 1.00 12.42 O ATOM 3199 O HOH X 320 9.426 13.453 65.971 1.00 9.90 O ATOM 3202 O HOH X 322 31.148 2.318 46.180 1.00 13.63 O ATOM 3205 O HOH X 324 13.896 24.311 51.333 1.00 12.28 O ATOM 3208 O HOH X 325 40.919 −4.284 28.002 1.00 15.70 O ATOM 3211 O HOH X 328 11.597 3.904 58.174 1.00 15.64 O ATOM 3214 O HOH X 329 7.013 21.318 64.262 1.00 17.19 O ATOM 3217 O HOH X 330 36.700 16.628 35.841 1.00 12.75 O ATOM 3220 O HOH X 331 35.919 8.860 52.061 1.00 14.21 O ATOM 3223 O HOH X 333 33.936 13.026 68.542 1.00 15.84 O ATOM 3226 O HOH X 334 28.439 18.984 38.377 1.00 15.56 O ATOM 3229 O HOH X 335 38.475 17.080 45.414 1.00 9.88 O ATOM 3232 O HOH X 336 29.059 23.743 65.349 1.00 13.08 O ATOM 3235 O HOH X 337 35.124 6.275 51.954 1.00 13.92 O ATOM 3238 O HOH X 338 7.219 2.126 57.698 1.00 19.91 O ATOM 3241 O HOH X 339 9.763 14.968 45.726 1.00 14.47 O ATOM 3244 O HOH X 340 13.387 8.617 69.453 1.00 16.22 O ATOM 3247 O HOH X 341 10.796 17.916 45.998 1.00 16.33 O ATOM 3250 O HOH X 342 18.031 18.962 65.202 1.00 15.58 O ATOM 3253 O HOH X 344 36.069 5.290 49.609 1.00 17.02 O ATOM 3256 O HOH X 345 29.619 18.922 35.833 1.00 22.31 O ATOM 3259 O HOH X 346 24.143 18.471 35.696 1.00 19.85 O ATOM 3262 O HOH X 348 21.129 −1.803 49.194 1.00 17.90 O ATOM 3265 O HOH X 349 38.093 13.627 38.055 1.00 14.45 O ATOM 3268 O HOH X 350 10.253 11.006 40.462 1.00 18.46 O ATOM 3271 O HOH X 351 10.793 13.130 44.082 1.00 22.46 O ATOM 3274 O HOH X 352 13.603 19.738 71.429 1.00 20.67 O ATOM 3277 O HOH X 353 7.557 20.765 60.119 1.00 17.34 O ATOM 3280 O HOH X 354 13.586 4.527 59.705 1.00 20.41 O ATOM 3283 O HOH X 355 37.530 2.113 41.859 1.00 24.82 O ATOM 3286 O HOH X 356 16.469 24.270 60.869 1.00 23.54 O ATOM 3289 O HOH X 357 29.936 18.089 32.080 1.00 26.88 O ATOM 3292 O HOH X 358 19.795 3.083 33.799 1.00 28.78 O ATOM 3295 O HOH X 359 38.291 20.499 44.138 1.00 22.25 O ATOM 3298 O HOH X 360 20.041 0.614 52.967 1.00 18.78 O ATOM 3301 O HOH X 361 29.369 0.094 63.771 1.00 21.56 O ATOM 3304 O HOH X 362 29.228 14.780 30.295 1.00 16.98 O ATOM 3307 O HOH X 363 28.984 12.621 69.804 1.00 21.48 O ATOM 3310 O HOH X 364 24.115 11.918 68.606 1.00 18.14 O ATOM 3313 O HOH X 365 37.961 6.598 47.868 1.00 16.76 O ATOM 3316 O HOH X 366 34.891 20.347 52.378 1.00 22.38 O ATOM 3319 O HOH X 367 31.698 28.109 59.447 1.00 28.93 O ATOM 3322 O HOH X 368 21.261 19.711 38.455 1.00 28.25 O ATOM 3325 O HOH X 369 30.748 20.856 45.143 1.00 28.82 O ATOM 3328 O HOH X 370 33.678 19.116 43.639 1.00 19.55 O ATOM 3331 O HOH X 371 25.018 10.141 66.819 1.00 19.92 O ATOM 3334 O HOH X 372 26.928 24.827 67.182 1.00 28.42 O ATOM 3337 O HOH X 373 32.538 19.487 36.343 1.00 21.66 O ATOM 3340 O HOH X 374 17.229 0.902 47.869 1.00 22.42 O ATOM 3343 O HOH X 375 19.360 15.924 71.231 1.00 27.13 O ATOM 3346 O HOH X 376 26.441 −6.252 39.178 1.00 25.00 O ATOM 3349 O HOH X 377 39.699 7.545 31.593 1.00 20.60 O ATOM 3352 O HOH X 378 7.020 9.932 60.846 1.00 28.81 O ATOM 3355 O HOH X 379 32.445 −1.887 56.117 1.00 29.10 O ATOM 3358 O HOH X 380 10.504 −0.374 38.640 1.00 31.66 O ATOM 3361 O HOH X 381 38.643 −4.052 56.330 1.00 28.25 O ATOM 3364 O HOH X 382 20.319 10.149 30.622 1.00 28.71 O ATOM 3367 O HOH X 383 16.249 21.410 43.896 1.00 33.25 O ATOM 3370 O HOH X 384 27.044 18.832 73.411 1.00 26.07 O ATOM 3373 O HOH X 385 17.653 5.995 63.119 1.00 20.40 O ATOM 3376 O HOH X 386 20.667 25.167 57.089 1.00 26.80 O ATOM 3379 O HOH X 387 24.565 25.212 44.311 1.00 27.12 O ATOM 3382 O HOH X 388 13.295 12.403 71.678 1.00 23.39 O ATOM 3385 O HOH X 389 31.967 −3.677 46.437 1.00 20.85 O ATOM 3388 O HOH X 400 23.013 1.080 36.088 1.00 20.68 O ATOM 3391 O HOH X 401 13.803 12.839 31.578 1.00 47.10 O ATOM 3394 O HOH X 402 35.493 −3.707 50.617 1.00 28.70 O ATOM 3397 O HOH X 403 11.319 24.471 65.742 1.00 31.09 O ATOM 3400 O HOH X 404 6.899 22.877 47.846 1.00 28.38 O ATOM 3403 O HOH X 405 40.035 11.449 34.241 1.00 29.40 O ATOM 3406 O HOH X 407 33.019 22.565 50.833 1.00 18.50 O ATOM 3409 O HOH X 410 19.785 18.612 72.020 1.00 35.87 O ATOM 3412 O HOH X 411 42.301 4.024 31.951 1.00 16.71 O ATOM 3415 O HOH X 412 3.955 5.909 56.974 1.00 32.26 O ATOM 3418 O HOH X 413 13.763 3.826 37.534 1.00 27.73 O ATOM 3421 O HOH X 414 31.801 −2.267 34.382 1.00 39.99 O ATOM 3424 O HOH X 415 15.515 6.216 61.471 1.00 27.52 O ATOM 3427 O HOH X 416 15.020 24.864 64.030 1.00 36.11 O ATOM 3430 O HOH X 417 12.455 5.600 62.319 1.00 24.57 O ATOM 3433 O HOH X 419 25.773 23.290 42.244 1.00 41.56 O ATOM 3436 O HOH X 420 41.722 7.186 39.109 1.00 29.19 O ATOM 3439 O HOH X 421 25.130 3.183 30.595 1.00 30.39 O ATOM 3442 O HOH X 422 37.744 3.789 58.380 1.00 40.22 O ATOM 3445 O HOH X 423 22.867 0.681 61.281 1.00 39.84 O ATOM 3448 O HOH X 424 42.153 6.744 60.740 1.00 23.29 O ATOM 3451 O HOH X 426 20.288 8.153 38.618 1.00 9.57 O ATOM 3454 O HOH X 427 35.253 14.363 31.838 1.00 50.83 O ATOM 3457 O HOH X 428 34.212 1.381 64.196 1.00 27.07 O ATOM 3460 O HOH X 429 14.434 1.392 52.833 1.00 22.25 O ATOM 3463 O HOH X 430 22.194 26.212 52.485 1.00 32.80 O ATOM 3466 O HOH X 432 9.238 16.975 65.774 1.00 13.96 O ATOM 3469 O HOH X 433 11.593 1.653 53.574 1.00 16.99 O ATOM 3472 O HOH X 434 14.051 4.174 43.951 1.00 20.14 O ATOM 3475 O HOH X 435 7.175 15.078 45.065 1.00 18.73 O ATOM 3478 O HOH X 437 11.480 1.805 56.261 1.00 18.06 O ATOM 3481 O HOH X 438 23.385 26.963 58.403 1.00 21.12 O ATOM 3484 O HOH X 440 7.211 20.077 47.511 1.00 26.15 O ATOM 3487 O HOH X 441 9.162 23.023 59.414 1.00 22.52 O ATOM 3490 O HOH X 442 32.407 4.550 28.277 1.00 23.52 O ATOM 3493 O HOH X 443 40.924 7.587 58.411 1.00 23.57 O ATOM 3496 O HOH X 444 9.907 2.424 59.932 1.00 32.11 O ATOM 3499 O HOH X 445 10.243 10.245 44.133 1.00 24.03 O ATOM 3502 O HOH X 446 34.992 21.525 48.756 1.00 23.33 O ATOM 3505 O HOH X 447 38.379 9.025 29.474 1.00 21.23 O ATOM 3508 O HOH X 449 16.335 20.049 71.265 1.00 25.31 O ATOM 3511 O HOH X 450 9.418 0.204 57.103 1.00 30.20 O ATOM 3514 O HOH X 451 20.747 17.031 31.596 1.00 31.73 O ATOM 3517 O HOH X 452 19.502 −0.547 41.887 1.00 24.23 O ATOM 3520 O HOH X 453 37.135 14.588 33.760 1.00 29.69 O ATOM 3523 O HOH X 454 26.888 0.464 64.181 1.00 34.17 O ATOM 3526 O HOH X 456 32.979 −4.890 37.436 1.00 35.49 O ATOM 3529 O HOH X 457 14.283 20.600 45.705 1.00 29.63 O ATOM 3532 O HOH X 458 26.230 25.153 53.647 1.00 27.48 O ATOM 3535 O HOH X 459 34.816 18.735 35.080 1.00 27.23 O ATOM 3538 O HOH X 460 4.994 3.925 56.064 1.00 37.00 O ATOM 3541 O HOH X 461 17.110 19.736 41.865 1.00 40.25 O ATOM 3544 O HOH X 463 22.791 −0.147 33.599 1.00 29.38 O ATOM 3547 O HOH X 465 14.549 −2.299 48.175 1.00 34.82 O ATOM 3550 O HOH X 466 33.884 12.558 26.982 1.00 30.87 O ATOM 3553 O HOH X 467 36.830 1.795 39.777 1.00 44.80 O ATOM 3556 O HOH X 468 18.656 26.477 68.955 1.00 45.51 O ATOM 3559 O HOH X 469 17.163 13.979 34.729 1.00 26.64 O ATOM 3562 O HOH X 470 44.148 4.270 41.881 1.00 25.80 O ATOM 3565 O HOH X 471 14.388 26.009 47.237 1.00 26.58 O ATOM 3568 O HOH X 472 29.188 25.033 52.227 1.00 38.08 O ATOM 3571 O HOH X 473 26.480 9.255 26.925 1.00 32.77 O ATOM 3574 O HOH X 474 15.463 24.100 71.378 1.00 33.75 O ATOM 3577 O HOH X 475 16.379 0.644 52.055 1.00 36.71 O ATOM 3580 O HOH X 476 12.848 −3.104 45.737 1.00 48.73 O ATOM 3583 O HOH X 478 26.768 27.292 66.187 1.00 31.72 O ATOM 3586 O HOH X 479 31.105 16.617 29.947 1.00 36.25 O ATOM 3589 O HOH X 480 19.085 25.365 71.424 1.00 54.75 O ATOM 3592 O HOH X 483 6.403 7.218 63.722 1.00 33.40 O ATOM 3595 O HOH X 484 34.676 −2.504 31.342 1.00 39.43 O ATOM 3598 O HOH X 485 29.761 6.193 26.274 1.00 33.63 O ATOM 3601 O HOH X 486 19.273 −0.131 49.941 1.00 49.50 O ATOM 3604 O HOH X 487 41.059 −2.510 31.457 1.00 30.75 O ATOM 3607 O HOH X 488 36.672 −0.693 42.753 1.00 29.42 O ATOM 3610 O HOH X 489 31.910 −3.375 54.078 1.00 24.32 O ATOM 3613 O HOH X 491 41.593 2.804 48.900 1.00 33.83 O ATOM 3616 O HOH X 492 36.681 0.663 35.856 1.00 27.43 O ATOM 3619 O HOH X 494 37.176 7.827 22.494 1.00 16.75 O ATOM 3622 O HOH X 495 14.784 3.915 46.392 1.00 17.06 O ATOM 3625 O HOH X 496 21.591 24.931 45.260 1.00 18.11 O ATOM 3628 O HOH X 497 29.412 27.494 64.245 1.00 26.46 O ATOM 3631 O HOH X 499 26.054 6.842 24.123 1.00 30.10 O ATOM 3634 O HOH X 500 19.812 24.826 43.219 1.00 31.66 O ATOM 3637 O HOH X 501 9.992 −0.590 53.205 1.00 34.87 O ATOM 3640 O HOH X 503 25.372 28.861 56.971 1.00 39.89 O ATOM 3643 O HOH X 504 17.053 16.002 37.141 1.00 32.34 O ATOM 3646 O HOH X 505 4.460 2.108 53.997 1.00 38.92 O ATOM 3649 O HOH X 506 26.465 17.162 75.469 1.00 43.82 O ATOM 3652 O HOH X 507 10.317 5.334 36.846 1.00 32.29 O ATOM 3655 O HOH X 508 9.711 2.947 49.218 1.00 26.65 O ATOM 3658 O HOH X 509 26.552 −4.697 49.755 1.00 49.34 O ATOM 3661 O HOH X 510 18.594 3.807 59.418 1.00 38.30 O ATOM 3664 O HOH X 511 28.435 −4.434 38.700 1.00 32.57 O ATOM 3667 O HOH X 512 46.206 0.057 55.972 1.00 47.60 O ATOM 3670 O HOH X 513 16.994 −2.195 41.625 1.00 45.55 O ATOM 3673 O HOH X 514 17.660 11.735 25.478 1.00 45.97 O ATOM 3676 O HOH X 515 26.466 12.186 74.746 1.00 57.29 O ATOM 3679 O HOH X 516 34.393 18.813 32.507 1.00 42.19 O ATOM 3682 O HOH X 517 11.225 13.884 36.104 1.00 37.70 O ATOM 3685 O HOH X 518 31.412 −0.234 65.532 1.00 44.80 O ATOM 3688 O HOH X 519 24.899 11.748 28.168 1.00 41.55 O ATOM 3691 O HOH X 520 11.219 15.023 41.393 1.00 49.39 O ATOM 3694 O HOH X 522 30.331 30.312 61.765 1.00 49.80 O ATOM 3697 O HOH X 523 36.831 −2.809 45.024 1.00 46.29 O ATOM 3700 O HOH X 524 36.482 12.740 67.772 1.00 33.34 O ATOM 3703 O HOH X 525 28.208 9.531 24.690 1.00 35.59 O ATOM 3706 O HOH X 526 13.689 0.204 57.175 1.00 29.37 O ATOM 3709 O HOH X 527 46.067 15.993 38.976 1.00 43.28 O ATOM 3712 O HOH X 528 25.883 −6.044 44.128 1.00 37.94 O ATOM 3715 O HOH X 530 20.809 26.042 47.566 1.00 39.73 O ATOM 3718 O HOH X 531 12.991 18.432 44.852 1.00 28.36 O ATOM 3721 O HOH X 532 28.964 −5.330 36.267 1.00 38.90 O ATOM 3724 O HOH X 533 22.960 2.218 31.945 1.00 33.89 O ATOM 3727 O HOH X 535 36.178 20.832 44.780 1.00 37.51 O ATOM 3730 O HOH X 536 4.574 1.902 51.421 1.00 46.14 O ATOM 3733 O HOH X 537 17.708 5.431 57.443 1.00 32.53 O ATOM 3736 O HOH X 538 32.827 13.445 73.510 1.00 38.62 O ATOM 3739 O HOH X 540 34.720 −2.566 29.023 1.00 41.06 O ATOM 3742 O HOH X 541 11.949 −2.854 42.648 1.00 48.23 O ATOM 3745 O HOH X 542 19.770 −0.101 33.503 1.00 59.26 O ATOM 3748 O HOH X 544 14.020 10.058 71.503 1.00 34.39 O ATOM 3751 O HOH X 545 14.076 4.892 28.832 1.00 50.07 O ATOM 3754 O HOH X 546 35.926 0.765 62.178 1.00 44.69 O ATOM 3757 O HOH X 548 7.514 2.581 49.864 1.00 60.69 O ATOM 3760 O HOH X 549 7.765 6.113 66.451 1.00 47.34 O ATOM 3763 O HOH X 550 23.019 26.499 48.819 1.00 38.93 O ATOM 3766 O HOH X 551 25.047 −0.668 28.874 1.00 61.87 O ATOM 3769 O HOH X 552 7.206 −0.125 52.753 1.00 47.97 O ATOM 3772 O HOH X 553 2.489 5.354 58.881 1.00 48.36 O ATOM 3775 O HOH X 555 24.539 −3.993 32.641 1.00 40.61 O ATOM 3778 O HOH X 556 39.684 −2.379 33.798 1.00 47.82 O ATOM 3781 O HOH X 557 27.988 23.583 70.986 1.00 45.95 O ATOM 3784 O HOH X 558 16.259 25.981 51.761 1.00 27.10 O ATOM 3787 O HOH X 559 13.222 −0.928 53.311 1.00 63.13 O ATOM 3790 O HOH X 560 20.224 29.108 69.260 1.00 57.89 O ATOM 3793 O HOH X 561 18.467 22.263 41.573 1.00 43.15 O ATOM 3796 S SO4 Y 601 35.678 12.996 56.623 0.70 7.67 S ATOM 3797 O1 SO4 Y 601 36.139 12.078 55.585 0.70 13.89 O ATOM 3798 O2 SO4 Y 601 35.797 12.210 57.841 0.70 8.58 O ATOM 3799 O3 SO4 Y 601 34.374 13.573 56.380 0.70 16.69 O ATOM 3800 O4 SO4 Y 601 36.638 14.082 56.557 0.70 11.71 O ATOM 3801 S SO4 Y 602 28.397 23.856 45.015 0.70 24.73 O ATOM 3802 O1 SO4 Y 602 27.281 24.307 45.834 0.70 25.61 O ATOM 3803 O2 SO4 Y 602 28.429 22.409 44.818 0.70 22.84 O ATOM 3804 O3 SO4 Y 602 29.613 24.229 45.743 0.70 29.50 O ATOM 3805 O4 SO4 Y 602 28.371 24.551 43.749 0.70 28.60 O ATOM 3806 S SO4 Y 603 42.900 20.990 81.966 0.70 29.41 O ATOM 3807 O1 SO4 Y 603 43.188 21.619 80.689 0.70 23.17 O ATOM 3808 O2 SO4 Y 603 43.931 20.109 82.488 0.70 30.62 O ATOM 3809 O3 SO4 Y 603 41.665 20.213 81.898 0.70 31.55 O ATOM 3810 O4 SO4 Y 603 42.726 22.118 82.860 0.70 32.81 O ATOM 3811 S SO4 Y 604 34.328 18.651 77.072 0.70 36.68 S ATOM 3812 O1 SO4 Y 604 33.645 17.961 75.979 0.70 36.62 O ATOM 3813 O2 SO4 Y 604 34.229 17.813 78.264 0.70 37.58 O ATOM 3814 O3 SO4 Y 604 33.640 19.910 77.283 0.70 35.92 O ATOM 3815 O4 SO4 Y 604 35.749 18.899 76.842 0.70 32.21 O ATOM 3816 S SO4 Y 605 20.767 −0.638 38.261 0.70 22.29 S ATOM 3817 O1 SO4 Y 605 21.835 −1.312 37.474 0.70 17.77 O ATOM 3818 O2 SO4 Y 605 20.750 0.822 37.951 0.70 20.00 O ATOM 3819 O3 SO4 Y 605 20.968 −1.030 39.620 0.70 20.92 O ATOM 3820 O4 SO4 Y 605 19.500 −1.224 37.828 0.70 28.20 O ATOM 3821 S SO4 Y 606 29.483 −6.067 47.771 0.70 27.74 S ATOM 3822 O1 SO4 Y 606 30.669 −5.901 46.925 0.70 29.74 O ATOM 3823 O2 SO4 Y 606 29.713 −7.052 48.825 0.70 30.93 O ATOM 3824 O3 SO4 Y 606 28.393 −6.449 46.875 0.70 29.72 O ATOM 3825 O4 SO4 Y 606 29.111 −4.839 48.457 0.70 18.37 O ATOM 3826 S SO4 Y 607 39.989 16.559 37.193 0.70 24.82 S ATOM 3827 O1 SO4 Y 607 39.493 17.029 35.901 0.70 31.09 O ATOM 3828 O2 SO4 Y 607 40.305 15.142 37.088 0.70 26.22 O ATOM 3829 O3 SO4 Y 607 38.909 16.710 38.115 0.70 24.47 O ATOM 3830 O4 SO4 Y 607 41.271 17.168 37.535 0.70 23.92 O ATOM 3831 S SO4 Y 608 31.245 20.872 79.064 0.70 129.03 S ATOM 3832 O1 SO4 Y 608 30.482 20.410 77.908 0.70 128.90 O ATOM 3833 O2 SO4 Y 608 31.998 19.756 79.631 0.70 129.11 O ATOM 3834 O3 SO4 Y 608 30.330 21.401 80.071 0.70 129.13 O ATOM 3835 O4 SO4 Y 608 32.164 21.927 78.647 0.70 129.06 O ATOM 3836 O3 GOL A 1 20.441 14.331 40.631 1.00 11.20 O ATOM 3838 C3 GOL A 1 20.040 15.547 40.073 1.00 11.44 C ATOM 3841 C2 GOL A 1 19.516 16.587 41.042 1.00 13.43 C ATOM 3843 O2 GOL A 1 20.554 17.176 41.804 1.00 14.62 O ATOM 3845 C1 GOL A 1 18.490 16.011 42.030 1.00 14.51 C ATOM 3848 O1 GOL A 1 17.955 17.070 42.775 1.00 17.23 O ATOM 3850 O3 GOL A 2 37.747 20.476 54.065 1.00 40.41 O ATOM 3852 C3 GOL A 2 37.169 19.407 53.339 1.00 36.60 C ATOM 3855 C2 GOL A 2 38.227 18.479 52.776 1.00 32.19 C ATOM 3857 O2 GOL A 2 38.442 17.656 53.815 1.00 26.25 O ATOM 3859 C1 GOL A 2 39.572 19.037 52.365 1.00 29.78 C ATOM 3862 O1 GOL A 2 40.418 18.008 51.922 1.00 38.17 O

TABLE 1A IC₅₀ values of peptides and ephrin-B2 for inhibition of mouse ephrin-B2 alkaline phosphatase (AP) binding to immobilized mouse EphB4 ectodomain Fc fusion protein using ELISA binding assays. Inhibitor IC₅₀ ephrin-B2 Fc   9 nM Human ephrin-B2 monomer  10 nM TNYLFSPNGPIARAW  15 nM (TNYL-RAW; SEQ ID NO:1)* -NYLFSPNGPIARAW  40 nM (NYLF-RAW; SEQ ID NO:30) --YLFSPNGPIARAW  40 nM (YLFS-RAW; SEQ ID NO:31) ---LFSPNGPIARAW >10 μM (LFSP-RAW; SEQ ID NO:32) TNYLFSPNGPIA 150 μM (TNYL; SEQ ID NO:33) TNYLFSPNGPIAGSGSK-biotin  50 μM (TNYL-biotin; SEQ ID NO:34) *Ephrin-B2 G-H loop: KFQEFSPNLWGLEFQK (SEQ ID NO:35)

TABLE 1B Binding of peptides and human ephrin-B2 to human EphB4 ephrin-binding domain. Ligand K_(d) (nM)* ΔG (kcal mol⁻¹) ΔH (kcal mol⁻¹) TΔS (kcal mol⁻¹) TNYL-RAW 71 ± 14 −9.8 ± 0.1 −14.7 ± 0.2 −4.9 ± 0.2 NYLF-RAW 65 ± 7  −9.8 ± 0.1 −15.5 ± 0.1 −5.7 ± 0.1 YLFS-RAW 80 ± 36 −9.7 ± 0.2 −13.8 ± 0.5 −4.1 ± 0.4 LFSP-RAW 3,500 ± 680   −7.4 ± 0.1  −5.3 ± 0.5  2.1 ± 0.4 TNYL ≧140,000 ND  −9.6 ± 0.3 ND ephrin-B2 40 ± 20 −10.2 ± 0.3   3.3 ± 0.1 13.4 ± 0.4 Experiments were performed at 25° C. in 50 mM Tris pH 7.8, 150 mM NaCl, 1 mM CaCl₂. All values (except for TNYL) represent the average of at least two experiments. *The K_(d) value for the TNYL peptide is a lower limit assuming a stoichiometry of 1 and at least 70% saturation of binding at a final peptide concentration of 300 μM.

TABLE 2 Crystallographic Statistics for the EphB4-TNYL-RAW Complex Resolution (Å)¹ 40-1.65 (1.71-1.65) Space Group P4₁2₁2 Unit Cell Dimensions (Å) a = b = 60.97, c = 151.7 Completeness (%) 100 (99.7) R_(sym) (%)² 3.9 (20.8) I/σ 42.7 (7.2) Mean Redundancy 3.7 (3.2) No. Reflections 32,786 R_(cryst) (%)³ 16.0 (17.4) R_(free) (%)⁴ 19.1 (20.2) R.m.s. deviations Bond length (Å) 0.02 Bond angle (°) 1.7 Improper (°) 1.4 Number of atoms Protein 1486 Solvent 214 Peptide 115 Sulfate 8 Glycerol 3 ¹Number in parentheses is for the highest shell. ²R_(sym) = |I I|/I, where I is the observed intensity and I is the average intensity of multiple symmetry-related observations of that reflection. ³R_(cryst) = F_(obs)| |F_(calc)/|F_(obs)|, where F_(obs) and F_(calc) are the observed and calculated structure factors. R_(sym) = |I I|/I, where I is the observed intensity and I is the average intensity of multiple symmetry-related observations of that reflection. ⁴R_(free) = F_(obs)| |F_(calc)/|F_(obs)| for 10% of the data not used at any stage of structural refinement.

EXAMPLES

Aspects of the present teachings may be further understood in light of the following examples, which should not be construed as limiting the scope of the present teachings in any way.

Example 1

Construct design, expression and purification of EphB4: Twelve sequential 4 amino acid truncations in human EphB4 were designed based on EphB4-EphB2 sequence alignment in the region C-terminal to the last β-strand in the EphB2 structure. The resulting fragments were cloned into the insect cell expression vector pBAC6 (Novagen, WI) under control of the heterologous GP64 signal peptide and containing a N-terminal six histidine tag. Constructs were sequence verified, and baculovirus was generated using homologous recombination into Sapphire Baculovirus DNA (Orbigen, CA) using the manufacturers protocol. After 3 rounds of viral amplification, a small scale expression screen was conducted for all constructs in both Sf9 and Hi5 insect cells. Briefly, 5E10+6 cells were infected with baculovirus at an MOI of 2 in 38 mm tissue culture dishes; cells were harvested at 48 hours post infection and supernatant containing secreted EphB4 was concentrated 10-fold and buffer exchanged into 50 mM Tris pH 7.8, 400 mM NaCl, and 5 mM imidazole using an Amicon Ultra 5K concentrator (Millipore, MA). The secreted protein was bound to Ni-NTA magnetic beads (Qiagen, CA), washed with 50 mM Tris pH7.8, 400 mM NaCl, 20 mM Imidazole buffer and eluted with 50 mM Tris pH 7.8, 400 mM NaCl, 250 mM Imidazole. Based on analysis of immobilized metal affinity chromatography (IMAC) elutes, the EphB4 (17-196) construct was identified as the highest expressor at ˜6 mg/L in Hi5 insect cells. Large scale expression was conducted using Wave Bioreactors(Wave Biotech LLC, NJ) at a MOI of 2 for 48 hours in Hi5 insect cells. Media containing secreted EphB4 was concentrated and buffer exchanged using a Hydrosart Crossflow filter (Sartorius, NY). Following IMAC purification on ProBond resin (Invitrogen, CA) as described above, EphB4 was concentrated to 5 mg/ml and loaded on a Superdex 75 16/60 column (GE HealthCare, NY). A small amount of aggregated material was removed by preparative size exclusion chromatography, while most of the sample eluted in a single peak corresponding to an EphB4 (17-196) monomer. The complete removal of the GP64secretion sequence and protein identity were confirmed by MALDI analysis.

Example 2

Crystallization: Purified EphB4 was concentrated to 10 mg/mL in 25 mM Tris, pH 7.8, 150 mM NaCl, and 5 mM CaCl2 in the presence of a 3-fold molar excess of TNYL-RAW peptide (SEQ ID NO: 1; Biopeptide, Inc.). The EphB4 17-196 construct was crystallized by sitting drop vapor diffusion at 20° C. against a reservoir of 2.2 M ammonium sulfate and 200 mM NaCl, and cryoprotected in 25% glycerol.

Example 3

Structure Determination: Crystals of the EphB4-TNYL complex grew in the P41212 space group (a=60.92, c=151.93). A single crystal diffracted to 1.65 Å resolution at 100 K on beamline 5-1 at the Advanced Light Source (Berkeley, Calif.), and were integrated, reduced and scaled using HKL2000 (Otwinowski, 1997). The structure was determined by molecular replacement with MolRep (CCP4i) (CCP4, 1994; Vagin, 1997) using the structure of apo EphB2 (pdb id:1NUK (Himanen et al., 1998)) as a search model. The structure was refined with CNS using torsion angle dynamics and the maximum likelihood function target (Table 2), and manual model building performed with the program O (Bringer et al., 1998; Jones et al., 1991). Electron density for the TNYL-RAW peptide was clear after the first round of refinement, with the positioning of the critical RAW sequence clearly evident in the initial |Fobs|−|Fcalc| maps (FIG. 2). The peptide was initially built as a polyalanine chain, while unbiased electron density for the peptide from simulated annealing omit maps was used to build the full peptide. Residues 17-196 from EphB4, and 14 of 15 residues from the TNYL-RAW peptide, could be readily traced into electron density. The final structure exhibits good geometry with no Ramachandran outliers. Figures were created with PyMol, Molscript, Raster3D, Dino, and Povray (DeLano, 2002; Esnouf, 1997;Kraulis, 1991; Merritt and Murphy, 1994).

Example 4

Isothermal titration calorimetry and ELISA experiments: EphB4and ephrin-B2 were either dialyzed or buffer exchanged into 50 mM Tris-Cl (pH 7.8 at 25° C.), 150 mM NaCl, 1 mM CaCl₂, prior to use in calorimetry experiments. Peptides were dissolved into the same buffer used for the dialysis of EphB4. The concentration of EphB4, ephrin-B2 and the peptides was determined by measuring the A₂₈₀ and using the theoretical-extinction coefficient (Gill and von Hippel, 1989). ITC experiments were performed with a Microcal MCS ITC at 25° C. Following an initial injection of 2 μl, titrations were performed by making 20 13 μl injections of peptide into EphB4 in the sample cell to produce an approximate final 2:1 ratio of injectant to sample in the cell. For most titrations the sample cell contained 15 μM EphB4 and the injection syringe contained a 200 μM solution of the peptide. Titrations with ephrin-B2 contained 13 μM EphB4 in the sample cell and 290 μM ephrin-B2 in the syringe. Prior to loading the sample cell, EphB4 was centrifuged at 18,000 g for 5 min at 4° C. to remove aggregates and degassed for 5 minutes at room temperature. Corrections for heats of dilution for the peptides and ephrin-B2 were determined by performing titrations of peptide or ephrin-B2 solutions into buffer. Dilution data were fit to a line and subtracted from the corresponding titration data. Titration data were analyzed using Origin ITC software (Version 5.0, Microcal Software Inc.) and curves were fit to a single binding site model (Wiseman et al., 1989). The low affinity of the TNYL peptide and the limited availability of EphB4 (17-196) precluded accurate determination of the K_(d) for this interaction by ITC. A lower limit for the binding constant was determined by performing a titration in which the sample cell contained 30 μM EphB4 and the injection syringe contained a 1.45 mM solution of the peptide, producing a final ratio of peptide to EphB4 of 10:1. The data was fit assuming a stoichiometry of 1 and at least 60% saturation of binding at the final peptide concentration (Turnbull and Daranas, 2003).

The ability of peptides to compete the binding of mouse ephrin-B2 alkaline phosphatase to immobilized mouse EphB4-Fc-His (R&D Systems) was measured by ELISA as previously described (Koolpe et al., 2005).

Other Embodiments

The detailed description set-forth above is provided to aid those skilled in the art in practicing the present invention. However, the invention described and claimed herein is not to be limited in scope by the specific embodiments herein disclosed because these embodiments are intended as illustration of several aspects of the invention. Any equivalent embodiments are intended to be within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description which do not depart from the spirit or scope of the present inventive discovery. Such modifications are also intended to fall within the scope of the appended claims.

REFERENCES CITED

All publications, patents, patent applications and other references cited in this application are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application or other reference was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Citation of a reference herein shall not be construed as an admission that such is prior art to the present invention. Publications incorporated herein by reference in their entirety include:

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1. A method for designing a drug which interferes with an activity of an EphB4 receptor, the method comprising: (a) providing on a digital computer a three-dimensional structure of a receptor-ligand complex comprising the EphB4 receptor and at least one ligand of the EphB4 receptor; and (b) using software comprised by the digital computer to design a chemical compound which is predicted to bind to the EphB4 receptor.
 2. A method according to claim 1, further comprising: (c) synthesizing the chemical compound; and (d) evaluating the chemical compound for an ability to interfere with an activity of the EphB4 receptor.
 3. A method according to claim 1, wherein the chemical compound is designed by computational interaction with reference to a three-dimensional site of the structure of the receptor-ligand complex, wherein the three-dimensional site is selected from the group consisting of EphB4 D-E and J-K loops.
 4. A method according to claim 3, wherein the three-dimensional site comprises Leu-48, Cys-61, Leu-95, Ser-99 Leu-100, Pro-101, Thr-147, Lys-149, Ala-155, and Cys-184 of SEQ ID NO:
 27. 5. A method according to claim 1, wherein the EphB4 receptor is a human EphB4 receptor.
 6. A method for determining a three-dimensional structure of a target EphB receptor-ligand complex structure comprising: (a) providing an amino acid sequence of a target EphB structure, wherein the three-dimensional structure of the target EphB structure is not known; (b) predicting a pattern of folding of the amino acid sequence in a three-dimensional conformation using a fold recognition algorithm; and (c) comparing the pattern of folding of the target structure amino acid sequence with the three-dimensional structure of a known EphB4 receptor-ligand complex.
 7. A method in accordance with claim 6, wherein the EphB4 receptor comprises an amino acid sequence as set forth in SEQ ID NOs: 2 or
 3. 8. A method in accordance with claim 6, wherein the EphB4 receptor consists essentially of an amino acid sequence as set forth in SEQ ID NOs: 2 or
 3. 9. A method in accordance with claim 7, wherein the known receptor-ligand complex comprises a three-dimensional structure described by atomic coordinates that substantially conform to atomic coordinates set forth in Table
 1. 10. A method according to claim 6, wherein the EphB4 receptor is a human EphB4 receptor.
 11. A method for generating a model of a three-dimensional structure of an EphB ligand complex, the method comprising: (a) providing an amino acid sequence of a reference EphB4 polypeptide and an amino acid sequence of a target EphB comprised by the EphB-ligand complex; (b) identifying structurally conserved regions shared between the reference EphB4 amino acid sequence and the target EphB amino acid sequence; and (c) assigning atomic coordinates from the conserved regions to the target EphB ligand complex.
 12. A method in accordance with claim 11, wherein the EphB4 polypeptide comprises an amino acid sequence as set forth in SEQ ID NOs: 2 or
 3. 13. A method in accordance with claim 11, wherein the EphB4 polypeptide consists essentially of an amino acid sequence as set forth in SEQ ID NOs: 2 or
 3. 14. A method in accordance with claim 11, wherein the target EphB-ligand complex comprises a three-dimensional structure described by atomic coordinates that substantially conform to atomic coordinates set forth in Table
 1. 15. A method in accordance with claim 11, wherein the reference EphB4-ligand complex comprises a three-dimensional structure described by atomic coordinates that substantially conform to atomic coordinates set forth in Table
 1. 16. A method according to claim 11, wherein the EphB4 polypeptide is a human EphB4 polypeptide.
 17. A method for generating a model of a three-dimensional structure of an EphB receptor-ligand complex, the method comprising: (a) providing an amino acid sequence of a known EphB4 receptor in complex with at least one known ligand of the EphB4 receptor; (b) providing an amino acid sequence of a target EphB receptor in complex with at least one target ligand of the EphB receptor; (c) identifying structurally conserved regions shared between the known receptor ligand complex amino acid sequence and the target receptor-ligand complex amino acid sequence; and (d) assigning atomic coordinates of the conserved regions to the target receptor ligand complex.
 18. A method in accordance with claim 17, wherein the EphB4 receptor comprises an amino acid sequence as set forth in SEQ ID NOs: 2 or
 3. 19. A method in accordance with claim 17, wherein the EphB4 receptor consists essentially of an amino acid sequence as set forth in SEQ ID NOs: 2 or
 3. 20. A method according to claim 17, wherein the EphB4 receptor is a human EphB4 receptor.
 21. A method according to claim 17, wherein the known receptor-ligand complex comprises a three-dimensional structure described by atomic coordinates that substantially conform to Table
 1. 22. A crystal comprising an EphB4 ligand binding domain and a ligand.
 23. A crystal according to claim 22, wherein the EphB4 ligand binding domain is a polypeptide having a sequence of SEQ ID NOs: 2 or
 3. 24. A crystal according to claim 22, wherein the EphB4 ligand binding domain consists essentially of EphB4 D-E and J-K loops.
 25. A crystal according to claim 22, wherein the EphB4 ligand binding domain consists essentially of Leu-48, Cys-61, Leu-95, Ser-99 Leu-100, Pro-101, Thr-147, Lys-149, Ala-155, and Cys-184 of SEQ ID NO:
 27. 26. A crystal according to claim 22, wherein the EphB4 ligand binding domain is a human EphB4 ligand binding domain.
 27. A crystal in accordance with claim 22, wherein the ligand is ephrin-B2.
 28. A crystal according to claim 22, wherein the ligand comprises Phe-120, Pro-122, Leu-124, Trp-125, and Leu-127 of ephrin-B2.
 29. A crystal according to claim 22, wherein the ligand comprises sequence motif NxWxL, wherein x is any amino acid.
 30. A crystal in accordance with claim 22, wherein the ligand is a polypeptide having SEQ ID NO:
 1. 31. A crystal according to claim 22, wherein the ligand is a polypeptide elected from the group consisting of polypeptides having SEQ ID NO: 4 through SEQ ID NO:
 26. 32. A crystal according to claim 22, wherein the ligand is a polypeptide having at least 50% sequence identity to a polypeptide selected from the group consisting of polypeptides having SEQ ID NO: 1 and SEQ ID NO: 4 through SEQ ID NO:
 26. 33. A crystal according to claim 22, wherein the ligand is a polypeptide having at least 75% sequence identity to a polypeptide selected from the group consisting of polypeptides having SEQ ID NO: 1 and SEQ ID NO: 4 through SEQ ID NO:
 26. 34. A crystal according to claim 22, wherein the ligand is a polypeptide having at least 90% sequence identity to a polypeptide selected from the group consisting of polypeptides having SEQ ID NO: 1 and SEQ ID NO: 4 through SEQ ID NO:
 26. 35. A crystal in accordance with claim 22, wherein the crystal comprises space group P41212 so as to form a unit cell of dimensions a=60.97 Å, b=60.97 Å, and c=151.7 Å.
 36. A crystal comprising a polypeptide having SEQ ID NOs: 2 or 3 complexed with a ligand, wherein the crystal is sufficiently pure to determine atomic coordinates of the complex by X-ray diffraction to a resolution of about 1.65 Å.
 37. A crystal according to claim 36, wherein the ligand comprises Phe-120, ProDocket 122, Leu-124, Trp-125, and Leu-127 of ephrin-B2.
 38. A crystal according to claim 36, wherein the ligand comprises sequence motif NxWxL, wherein x is any amino acid.
 39. A crystal in accordance with claim 36, wherein the ligand is a polypeptide having SEQ ID NO:
 1. 40. A crystal according to claim 36, wherein the ligand is a polypeptide selected from the group consisting of polypeptides having SEQ ID NO: 4 through SEQ ID NO:
 26. 41. A crystal according to claim 36, wherein the ligand is a polypeptide having at least 50% sequence identity to a polypeptide selected from the group consisting of polypeptides having SEQ ID NO: 1 and SEQ ID NO: 4 through SEQ ID NO:
 26. 42. A crystal according to claim 36, wherein the ligand is a polypeptide having at least 75% sequence identity to a polypeptide selected from the group consisting of polypeptides having SEQ ID NO: 1 and SEQ ID NO: 4 through SEQ ID NO:
 26. 43. A crystal according to claim 36, wherein the ligand is a polypeptide having at least 90% sequence identity to a polypeptide selected from the group consisting of polypeptides having SEQ ID NO: 1 and SEQ ID NO: 4 through SEQ ID NO:
 26. 44. A polypeptide having SEQ ID NOs: 2 or 3 in complex with a ligand.
 45. A complex according to claim 44, wherein the ligand is an ephrin.
 46. A complex according to claim 45, wherein the ephrin is ephrin-B2.
 47. A complex according to claim 44, wherein the ligand comprises Phe-120, Pro-122, Leu-124, Trp-125, and Leu-127 of ephrin-B2.
 48. A complex according to claim 44, wherein the ligand comprises sequence motif NxWxL, wherein x is any amino acid.
 49. A complex according to claim 44, wherein the ligand is a polypeptide having SEQ ID NO:
 1. 50. A complex according to claim 44, wherein the ligand is a polypeptide selected from the group consisting of polypeptides having SEQ ID NO: 4 through SEQ ID NO:
 26. 51. A complex according to claim 44, wherein the ligand is a polypeptide having at least 50% sequence identity to a polypeptide selected from the group consisting of polypeptides having SEQ ID NO: 1 and SEQ ID NO: 4 through SEQ ID NO:
 26. 52. A complex according to claim 44, wherein the ligand is a polypeptide having at least 75% sequence identity to a polypeptide selected from the group consisting of polypeptides having SEQ ID NO: 1 and SEQ ID NO: 4 through SEQ ID NO:
 26. 53. A complex according to claim 44, wherein the ligand is a polypeptide having at least 90% sequence identity to a polypeptide selected from the group consisting of polypeptides having SEQ ID NO: 1 and SEQ ID NO: 4 through SEQ ID NO:
 26. 54. A therapeutic compound that inhibits an activity of an EphB4 receptor, wherein the compound is selected by a) performing a structure based drug design using a three-dimensional structure determined for a crystal comprising an EphB4 receptor and a ligand; b) contacting a sample comprising the EphB4 receptor with the compound, and c) detecting inhibition of at least one activity of the EphB4 receptor.
 55. A compound according to claim 54, wherein the EphB4 is a polypeptide having SEQ ID NOs: 2 or
 3. 56. A compound according to claim 54, wherein the EphB4 receptor is a human EphB4 receptor.
 57. A compound according to claim 54, wherein the ligand is a polypeptide having SEQ ID NO:
 1. 58. A compound according to claim 54, wherein the ligand is a polypeptide selected from the group consisting of polypeptides having SEQ ID NO: 4 through SEQ ID NO:
 26. 59. A compound according to claim 54, wherein the ligand is a polypeptide having at least 50% sequence identity to a polypeptide selected from the group consisting of polypeptides having SEQ ID NO: 1 and SEQ ID NO: 4 through SEQ ID NO:
 26. 60. A compound according to claim 54, wherein the ligand is a polypeptide having at least 75% sequence identity to a polypeptide selected from the group consisting of polypeptides having SEQ ID NO: 1 and SEQ ID NO: 4 through SEQ ID NO:
 26. 61. A compound according to claim 54, wherein the ligand is a polypeptide having at least 90% sequence identity to a polypeptide selected from the group consisting of polypeptides having SEQ ID NO: 1 and SEQ ID NO: 4 through SEQ ID NO:
 26. 62. A three-dimensional computer image of the three-dimensional structure of an EphB4-ligand complex, wherein the structure substantially conforms to the three-dimensional coordinates listed in Table
 1. 63. A computer-readable medium encoded with a set of three-dimensional coordinates set forth in Table 1, wherein, using a graphical display software program, the three-dimensional coordinates of Table 1 create an electronic file that can be visualized on a computer capable of representing said electronic file as a three-dimensional image.
 64. A computer-readable medium encoded with a set of three-dimensional coordinates of a three-dimensional structure which substantially conforms to the three-dimensional coordinates represented in Table 1, wherein, using a graphical display software program, the set of three-dimensional coordinates create an electronic file that can be visualized on a computer capable of representing said electronic file as a three-dimensional image.
 65. A method for assaying EphB4 receptor binding to a compound, the method comprising a) providing an EphB4 receptor bound with a polypeptide having SEQ ID NO: 1; b) contacting the ligand-bound EphB4 receptor with a compound; and c) detecting the release of the polypeptide having SEQ ID NO: 1 from the EphB4 receptor, wherein the release of the polypeptide having SEQ ID NO: 1 is indicative of the compound binding to the EphB4 receptor.
 66. A method according to claim 65, wherein the EphB4 receptor is a polypeptide having SEQ ID NOs: 2 or
 3. 67. A method according to claim 65, wherein the EphB4 receptor consists essentially of EphB4 D-E and J-K loops.
 68. A method according to claim 65, wherein the EphB4 receptor consists essentially of Leu-48, Cys-61, Leu-95, Ser-99 Leu-100, Pro-101, Thr-147, Lys-149, Ala-155, and Cys-184 of SEQ ID NO:
 27. 69. A method according to claim 65, wherein the EphB4 receptor is a human EphB4 receptor.
 70. A method for crystallizing an EphB4 receptor, the method comprising: a) providing an EphB4 receptor in contact with a first polypeptide having SEQ ID NO: 1; and b) contacting the EphB4 receptor in contact with the first polypeptide with a second polypeptide having at least 50% sequence identity to SEQ ID NO: 1, but not identical to SEQ ID NO: 1, wherein the EphB4 receptor in contact with the first and second polypeptides forms an EphB4 receptor crystal.
 71. A method according to claim 70, wherein the second polypeptide comprises at least 75% sequence identity to SEQ ID NO:
 1. 72. A method according to claim 70, wherein the second polypeptide comprises at least 90% sequence identity to SEQ ID NO:
 1. 73. A method for crystallizing an EphB4 receptor, the method comprising: a) providing an EphB4 receptor in contact with a polypeptide having SEQ ID NO: 1; and b) contacting the EphB4 receptor in contact with the polypeptide with a compound of claim 54, wherein the EphB4 receptor in contact with the polypeptide and the compound forms an EphB4 receptor crystal.
 74. A composition comprising EphB4 receptor, a ligand, and a compound of claim
 54. 75. A composition according to claim 74, wherein the EphB4 receptor is a polypeptide having SEQ ID NOs: 2 or
 3. 76. A composition according to claim 74, wherein the EphB4 receptor consists essentially of EphB4 D-E and J-K loops.
 77. A composition according to claim 74, wherein the EphB4 receptor consists essentially of Leu-48, Cys-61, Leu-95, Ser-99 Leu-100, Pro-101, Thr-147, Lys-149, Ala-155, and Cys-184 of SEQ ID NO:
 27. 78. A composition according to claim 74, wherein the EphB4 receptor is a human EphB4 receptor.
 79. A composition according to claim 74, wherein the ligand is a polypeptide having SEQ ID NO:
 1. 80. A composition according to claim 74, wherein the ligand is a polypeptide selected from the group consisting of polypeptides having SEQ ID NO: 4 through SEQ ID NO:
 26. 81. A composition according to claim 74, wherein the ligand is a polypeptide having at least 50% sequence identity to a polypeptide selected from the group consisting of polypeptides having SEQ ID NO: 1 and SEQ ID NO: 4 through SEQ ID NO:
 26. 82. A composition according to claim 74, wherein the ligand is a polypeptide having at least 75% sequence identity to a polypeptide selected from the group consisting of polypeptides having SEQ ID NO: 1 and SEQ ID NO: 4 through SEQ ID NO:
 26. 83. A composition according to claim 74, wherein the ligand is a polypeptide having at least 90% sequence identity to a polypeptide selected from the group consisting of polypeptides having SEQ ID NO: 1 and SEQ ID NO: 4 through SEQ ID NO:
 26. 